Genetic differentiation between and within five varieties of Euphrasia stricta (var. brevipila, var. gotlandica, var. stricta, var. suecica and var. tenuis) on Gotland was investigated, using amplified fragment length polymorphism, AFLP. The varieties are described in the literature by morphology and association to habitat type. We wanted to investigate whether the varieties are locally adapted populations to the typical habitat type for each variety or if they are preadapted to certain habitat types and have colonized Gotland in their present form. A constrained principal coordinate analysis revealed three genetically differentiated subunits within the species. The two early-flowering varieties suecica and tenuis each formed a distinct group, while the three late-flowering varieties brevipila, gotlandica and stricta together formed the third group. A phylogenetic tree confirms the partitioning into three groups. Within the group containing the late-flowering varieties there are populations that pair as each other's closest relatives, but belong to different varieties. These pairs are also geographically adjacent. The phylogenetic tree had a “star-like” appearance indicating a stronger divergence between populations than between varieties. The same pattern was seen in the partitioning of genetic diversity, with a lower amount of genetic variation occurring between varieties, F_ST=0.14, than between populations within the varieties, F_ST ranging from 0.26 to 0.60. In Euphrasia stricta the varieties suecica and tenuis and the group containing the varieties stricta/gotlandica/brevipila are likely to have a phylogeographical history outside Gotland, or an ancient and concealed local origin on the island. Within the group stricta/gotlandica/brevipila local evolutionary events seem to determine the variety identity, probably through local adaptation. Natural selection, genetic drift and mutations create genetic differentiation between populations. Gene flow, on the other hand, may counteract these processes (Slatkin 1987). Local adaptation is affected by the stability and strength of the natural selection and the amount of gene flow (Rice and Mack 1991, van Tienderen 1992, Miller and Fowler 1994), but also by the amount of genetic variability for the character that selection works on (Dudley 1996). Many studies have been done in the area of local adaptation (Lönn 1993, Prentice et al. 1995, Lönn et al. 1996, Liviero et al. 2002), and some studies have identified selective agents causing the adaptations. The selective agents are for example small differences in ecological niches and frequency dependent selection caused by pathogens (Parker 1994) or differences in selection regimes in different habitats (Kittelson and Maron 2001). Recent findings on ecological speciation emphasizes the importance of niche-shifting in local populations or groups of populations (Levin 2003) and rapid accumulation of beneficial mutations in isolated small populations (Rieseberg et al. 2003). Evolution works on different spatial and temporal scales, which makes it important to consider these different scales when studying evolutionary processes. Looking at the local phylogeny, geographic and temporal aspects are important when they link evolutionary processes to the extant landscape and the properties of the genetic structure. Linking evolutionary processes to the extant landscape is an important tool in evaluating evolutionary potential and predicting effects of landscape changes. Regional dynamics within species, using varieties/ecotypes, may also give information on initiation of speciation events. Manel et al. (2003) introduce and define landscape genetics as the combination of molecular population genetics and landscape ecology. The advantage of landscape genetics is the combination of the broad geographical span of scales (landscape to microclimate) and the high genetic resolution (individuals) compared to biogeography and phylogeography, which focuses more on species level at a broad spatial and temporal scale. Escudero et al. (2003), like Manel et al. (2003), put an emphasis on the spatial analysis of genetic diversity where a second step is to find ecological or demographic processes that could have shaped the genetic structure. A more direct approach is to measure habitat and genetic properties at many geographic locations and then model the biological processes shaping the spatial genetic structure (Lönn 1993, Prentice et al. 1995), which is the approach we intend to follow here. Molecular markers will be able to trace stochastic processes like drift and gene-flow (Page and Holmes 1998) as well as selective events through hitch-hiking (Hedrick 1980) and linkage events: AFLP has been used to identify quantitative trait loci by Via and Hawthorne (2002) and to explore the role of directional selection in whitefish ecotypes by Campbell and Bernatchez (2004). Yeo (1954, 1956, 1961, 1962, 1964, 1966, 1968) has done an extensive study of the cytology, hybridisation, cultivation, germination and relationship between species of British and European Euphrasia species. Yeo (1968) concludes that differences in chromosome number, habitat preferences and spatial distribution drives the speciation of Euphrasia in Europe and limits the hybridisation between species. The hybridisation may however result in new gene combinations for selection to work on (Yeo 1968). Yeo (1968) suggests that Euphrasia has gone through a fast and quite recent evolution in Europe after the last glaciation since Euphrasia has interfertile species of which many are endemic to small areas. Today, species differentiation within Euphrasia may be due to vegetation history, hybridisation and the parallel selection of well-adapted biotypes in similar or identical habitats (Karlsson 1976). Both Karlsson (1986) and Yeo (1968) put emphasis on the habitat specialization as an important factor in speciation referring to high morphological variability and hybridisation creating possibilities to evolve habitat specializations in Euphrasia.Zopfi (1998) showed in cultivation experiments that there is a genetic basis for different ecotypic variants of Euphrasiarostkoviana defined by grassland management, concerning onset of flowering, seed size and flowering period, life-history characters that are important adaptations to grazing and mowing. Euphrasia stricta is a tetraploid annual hemiparasite belonging to the Scrophulariaceae family (Yeo 1968, Krok and Almquist 2001). The species occurs all over Europe, except on the British Isles and in Spain and Portugal (Hultén and Fries 1986). In Sweden five varieties are found, which are subdivided based on morphology, phenology and habitat preference (Krok and Almquist 2001). They all grow on the Baltic island of Gotland, which is situated east of Sweden consisting of Silurian limestone (Fredén 1994). Euphrasia stricta var. suecica and E. stricta var. tenuis grow in traditionally managed wooded hay meadows and both are early flowering (Karlsson 1984). The variety suecica is red-listed according to the Swedish Red List (Gärdenfors 2000) and exists only in meadows on Gotland. The variety tenuis exists not only on Gotland but also on the Swedish mainland although it is declining throughout its distribution range. The populations of suecica and tenuis on Gotland are well known (Karlsson 1984, Petersson 1999). Euphrasia stricta var. stricta and E. stricta var. brevipila occur in pastures, along paths and on cultivated land. They flower later in the summer than var. suecica and var. tenuis. The variety stricta is common on the calcareous ground on Gotland with short grass turf, but rare on the mainland in contrast to the variety brevipila, which is common in whole of Sweden except on Gotland. The variety brevipila prefers soils that contain more sand compared to stricta. The late-flowering variety gotlandica is only found on Gotland and Öland, the second Baltic island on the Swedish east coast, were it is restricted to temporary pools on limestone ground (alvar) (Karlsson 1986). All Euphrasia species seems to lack a persistent seed bank (Karlsson 1984), but seeds have survived for up to three years in pots in cultivation experiments of other Euphrasia species (Yeo 1961). Artificial selfing and crosses within and between populations of Euphrasia stricta var. stricta yield high fertility in progeny pollen, 70–100% in between population crosses and 90–100% in selfing or within population crosses (Karlsson 1986). Flowering time for Euphrasia is not only dependent on habitat, but also to some extent on temperature and host attachment (Wilkins 1963, Yeo 1964, Molau 1993, Svensson et al. 2001, Svensson and Carlsson 2004). Euphrasia stricta probably have a mixed mating system (von Wettstein 1896). Based on these factors, which separates the varieties spatially and temporal, the aim of this study was to examine whether the varieties are locally adapted ecotypes that have evolved more than one time on the studied geographical scale or if they are distinct units over the region, implying colonization from outside or a single evolutionary event