ABSTRACT Arabidopsis lyrata ssp. lyrata is widespread but edaphically restricted, which suggests that the dispersal ability and level of genetic exchange among populations might be limited. We assessed levels of genetic diversity and population differentiation within and among six A. lyrata ssp. lyrata populations from along the eastern seaboard of the United States by examining variation at nine microsatellite loci among 233 individuals. These data were also evaluated to assess any relationship between geographic and genetic distance and correlation of genetic diversity with population size. Measures of genetic diversity across all 9 microsatellite loci were high in most populations. Genetic diversity, as measured by number of alleles, was generally higher in the Maryland and New York populations, as compared with the southern most sampled populations in Virginia. Genetic differentiation among regions was highly significant and large (FST 5 0.47). This was supported by several analyses, suggesting that there is little gene flow among the regions sampled. There was a strong positive correlation between geographic and genetic distance, which is indicative of the pattern of isolation-bydistance. Population size correlated with the number of alleles within populations, supporting population genetic theory that larger populations harbor greater genetic diversity. Wright’s FST and genetic structure analysis also provided evidence of genetic partitioning at small (,100 m) geographic distances between two areas sampled within one locality. These results indicate that the largest proportion of variation was explained by within population differences and may have implications for the conservation of this widespread but fragmented taxon.