O. acuminata var. jingxiensis, an endangered endemic species, is only distributed in rivers or streams in karst areas of southwest China. In this study, the genetic variation of 10 natural populations sampled from karst rivers was detected using 12 SSR markers. The results revealed a moderate level of genetic diversity within the populations (mean H_E = 0.441, I = 0.781) and population structure which significantly correlated with geographic distance (r = 0.825, P < 0.01). This pattern could be attributed to either restricted gene flow (N^_m = 0.341) between the long-isolated populations or the occurrence of biparental inbreeding in closely distributed individuals (F_rs = 0.362). The analysis of molecular variance (AMOVA) revealed a high level of genetic variation among the populations (F_rs = 0.423) of O. acuminata var. jingxiensis and accounted for over 55% of the total genetic differentiation. Compared to gene flow, genetic drift is more likely to promote the current genetic structure because of low gene exchange among isolated habitats. Complete geographical isolation offers an opportunity for allopatric speciation at relatively small spatial scales that promotes high endemism. Our result indicated that it is likely that cryptic varieties exist in O. acuminata, such as Baishou (BS) and Luzhai (LZ) populations. Based on the available results, both in situ and ex situ efforts should be used to conserve this species.