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Aphyocypris normalis (Teleostei: Cyprinidae) is an endemic species in South China, but little is known about its genetic structure. This study examined the population structure of A. normalis using sequences of the mitochondrial DNA control region and cytochrome b gene (2,086 bp). In total, 107 specimens were collected from nine populations. All 105 mtDNA haplotypes were identified as belonging to two allopatric phylogroups. The results of a statistical dispersal-vicariance analysis (S-DIVA) suggested that the ancestral populations of A. normalis were distributed widely on Hainan Island and east of the Leizhou Peninsula. A comparison of the fixation indices N-ST (0.532) and G(ST) (0.004) revealed that the phylogeny and geography had a significant relationship. Our study found that (1) the Wuzhishan and Yinggeling Mountain Range was an important barrier limiting gene exchange between populations on both sides; (2) cyclic climate changes may have shaped migrations and population differentiations; and (3) different colonization times caused different population diversities between codistributed species. In addition, the inter-and intraspecific diversities of the genus Aphyocypris were estimated.