Abstract: The Chishui River, the important part of the Rare and Endemic Fishes Nature Reserve in the upper Yangtze River basin, plays an irreplaceable role in biodiversity conservation. The present study used mitochondrial DNA (mtDNA) Cyt b gene as molecular maker to perform the genetic diversity and phylogeographic analyses of two species of Homatula (H. variegata and H. potanini) from the Chishui River and nearby rivers. 42 haplotypes were identified for 135 individuals of H. variegata from 5 rivers, and 12 haplotypes for 52 individuals of H. potanini from 3 rivers. Rela-tively high haplotype diversity were detected (h_{H. variegata}=0.936, h_{H. potanini}=0.821) with low nucleotide diversity in H. variegates (π=0.00493) and high nucleotide diversity in H. potanini (π=0.01105). Haplotype diversity and nucleotide diversity of H. variegates in the Chishui River were 0.891 and 0.00208, respectively, and their values were 0.64 and 0.00390 for H. potanini from the same river. Phylogenetic analyses for all haplotypes based on ML and BI methods revealed that H. variegata and H. potanini were separately clustered as their own monophy. The phylogeny of H. variegates clad showed the population from the Chishui River as paraphy located at the basal position of this clad, and all samples were separately clustered together according to their drainages with an obvious pattern of geographic distribution, while this pattern was not observed in samples of H. potanini. No haplotypes from the Chishui River were shared with other rivers, showing a significant isolation and difference in geographic distribution patterns. Because of the far distance between these rivers, it is speculated that the formation of this pattern was not caused by geological movement, but maybe resulted from the fluctuation of water level in the ancient time. Mismatch distribution analysis indicted the recent demographic expansions of Homatula populations in the Chishui River, but not all neutrality tests supported this hypothesis. These results suggest the relatively complex demographic history of Homatula species in the Chishui River.