To understand the demographic history of Arabidopsis thaliana within its native geographical range, we have studied its genetic structure in the Iberian Peninsula region. We have analyzed the amount and spatial distribution of A. thaliana genetic variation by genotyping 268 individuals sampled in 100 natural populations from the Iberian Peninsula. Analyses of 175 individuals from 7 of these populations, with 20 chloroplast and nuclear microsatellite loci and 109 common single nucleotide polymorphisms, show significant population differentiation and isolation by distance. In addition, analyses of one genotype from 100 populations detected significant isolation by distance over the entire Iberian Peninsula, as well as among six Iberian subregions. Analyses of these 100 genotypes with different model-based clustering algorithms inferred four genetic clusters, which show a clear-cut geographical differentiation pattern. On the other hand, clustering analysis of a worldwide sample showed a west-east Eurasian longitudinal spatial gradient of the commonest Iberian genetic cluster. These results indicate that A. thaliana genetic variation displays significant regional structure and consistently support the hypothesis that Iberia has been a glacial refugium for A. thaliana. Furthermore, the Iberian geographical structure indicates a complex regional population dynamics, suggesting that this region contained multiple Pleistocene refugia with a different contribution to the postglacial colonization of Europe.