Abstract: Spinibarbus caldwelli(Cyprinidae,Barbine,Spinibarbus)is a kind of small to middle sized fish dwelling in the middle or at the bottom of rivers,widely distributed in Yuanjiang River, Zhujiang River, Jiulongjiang River, Minjiang River, Changjiang River, Hainan and Taiwan. Because of the differences of ecological environments, morphological variations were found among the samples of S. caldwelli from different localities. In the present paper, with Spinibarbus sinensis as the outgroup, the cytochrome b genes(1140)of S. caldwelli specimens from different localities were sequenced to study biogeographical process of this species. Molecular phylogenetic trees were constructed according to the neighbor-joining method and the most parsimony method in Mega 2.0 software. The results showed that variations between samples from Changjiang and Zhujiang rivers, Changjiang and Minjiang rivers, Changjiang and Jiulongjiang rivers,were 1.2%-2.3%,2.7%-3.7%,and 3.1%-4.2% respectively. All these are much lower than the interspecies variations (13.2%-14.6%). These variations and molecular phylogenetic trees indicate that the biogeographical process might be that water systems in southeastern coast region separated from the inland water systems first, then separation between Zhujiang River and Changjiang River occurred,and these events should be in the Quaternary.