Genetic diversity within and among 42 native populations of Bromus tectorum (cheatgrass) was characterized within two regions, the eastern Mediterranean and the western Mediterranean. Two hypotheses were tested for the genetic diversity of these populations: (1) populations from the eastern Mediterranean are more genetically diverse compared with populations to the west, a potential consequence of the species' westward dispersal with the spread of agriculture, and (2) populations across the Mediterranean contain comparable genetic diversity but display high genetic differentiation, a potential consequence of both regions having served as refugia during glacial advances in the late Quaternary Period. Populations in the eastern Mediterranean possess 16 polymorphic loci and 37 multilocus genotypes. In contrast, populations from the western Mediterranean include a subset of these polymorphic loci (9) and fewer multilocus genotypes (19), consistent with the dispersal of B. tectorum with the east-west Holocene spread of agriculture. Among the 19 multilocus genotypes identified in populations from the western Mediterranean, 13 are undetected among eastern Mediterranean populations. Average genetic diversity within populations from the eastern Mediterranean is nonetheless comparable to the genetic diversity in populations from the Iberian Peninsula, whereas diversity is the lowest in the populations from southern France. Our results suggest a prominent role for agriculture in the grass's western spread, although glacial history and environmental heterogeneity also could have influenced the grass's genetic diversity. The exceptionally high level of self-pollination (>99%) in B. tectorum has contributed to preserving the genetic signature associated with the species' biogeographical history across the Mediterranean region.