Hemibarbus labeo and Hemibarbus medius (Cypriniformes:Cyprinidae:Gobioninae:Hemibarbus) are primary freshwater fish species, and their wide distribution makes them ideal models for phylogeographical studies. In this study, variations in the population genetics and phylogeographical patterns of 148 specimens belonging to 8 populations of H. labeo and 9 populations of H. medius collected from 17 drainage systems in south China were investigated using the nucleotide sequences of the mtDNA Cytochrome b gene (1140 bp). The results reveal 128 variable sites (11.23%) within the gene sequences of H. labeo and H. medius. A total of 41 haplotypes were identified, with the haplotype diversity (h) and nucleotide diversity (π) being 0.954 and 0.02153, respectively. This indicates a high level of genetic diversity and evolutionary potential in both species. The results of the neighbor-joining tree demonstrate that H. labeo and H. medius individuals fall into two major clades (clade I and clade II):clade I is composed of all specimens collected from the Hanjiang and Jiulongjiang Rivers, all of which were identified as H. labeo. Meanwhile, all remaining populations fell into clade II, which includes specimens of both H. labeo and H. medius. The genetic distance between clade I and clade II was 5.1%, while that between H. labeo and H. medius was 3.2%. Our results indicate that the specimens collected from these sampling localities may not be differentiated into two or more subspecies. The haplotype network indicates that the populations of the Hanjiang and Jiulongjiang Rivers exhibit a relatively high level of genetic variation compared to that of the rest of the rivers in the region, and that the populations from Hainan Island and Moyangjiang River were genetically close to those from the Pearl River System, but not to those from the Yangtze River System,Hanjiang River, Jiulongjiang River, Minjiang River, Oujiang River and Qiantangjiang River. The H. medius populations of south China seem to have originated from the Xijiang River and reached south China by one of two routes:one route may be through the Xijiang River to the Beijiang and Dongjiang rivers; and the other may be though the southern Guangxi drainages to Hainan Island, and then to the Moyangjiang and Beijiang rivers through the Qiongzhou Strait. According to the neighbor-joining tree and the haplotype network, five regions of genetic distribution were defined:Hainan Island and the Moyangjiang River region; the Pearl River region; the Yangtze River region; the Hanjiang and Jiulongjiang river region; and the Minjiang, Oujiang, and Qiantangjiang river region. An analysis of molecular variance (AMOVA) showed that the genetic variation of populations among regions was 54.50%; conversely, the genetic variation among populations within regions was 18.64%, and that within the populations of different regions was 26.86%. This indicates that the greatest genetic variation is found among the populations of the different regions. Mismatch distribution and tests of neutrality taking populations of H. labeo and H. medius into account both together and separately were all multimodal types, and the value of Fu, Li's D, and Tajima's D for all comparisons were all negative but non-significant or positive. The results reveal relatively stable populations of H. labeo and H. medius. Mismatch distribution and tests of neutrality also showed that both clade I and clade II were relatively stable; however, the population of the Changhuajiang River has undergone an obvious population expansion.