The pattern of genetic differentiation between diverging species receives much attention as one of the key observable features of speciation. It has often been suggested that introgression between closely related species occurs commonly where their distributions overlap, leading to their becoming more morphologically and genetically similar, but there are a few opposite results. However, most of these studies have been carried out with animals and separate species; few have looked at intraspecific cases, especially in plants. Here, we conduct a comparative study on patterns of genetic differentiation among populations of two varieties of Oreocharis benthamii in allopatry and sympatry based on ISSR data for 754 individuals from 26 populations, in order to understand the processes leading to speciation. Contrary to expectations, the facultative xenogamy (mixed mating) species O. benthamii has a relatively low genetic diversity within populations (H=0.1014, I=0.1528) and high genetic differentiation among populations (GST=0.5867, ФST=0.659), as is typically found for selfing species. Genetic variance between the two varieties in sympatric populations (44%, ФST=0.444) is significantly more than that in allopatric populations (14%, ФST=0.138). Consistent with the taxonomical delimitation of the two varieties, all sampled individuals of O. benthamii clustered into two genetic groups. Moreover, the genetic structures of populations of both varieties is correlated with their different geographical origins. Our studies show that significant divergence between sympatric populations of the two varieties could be attributed primally to reinforcement by genetic divergent selection in sympatry where secondary contact had occurred. The major proportion of the genetic variation in outcrossing and mixed mating plants may exist among populations when the populations are distributed in fragmented habitats, due to the paucity of suitable habitat combined with inefficient seed dispersal mechanism and limited pollinator foraging area that may limit the gene flow.