Geological events and landscape features, as well as changes in the climate during the Pliocene period, have shaped the distribution of genetic diversity and demographic history of freshwater fish in mainland China. In this study, we investigated the phylogeny and population genetic structure of Acrossocheilus species (A. fasciatus, A. kreyenbergii and A. wenchowensis) in the Zhejiang region by the mitochondrial cytochrome b (cyt-b) and control (D-loop) region s. Mitochondrial phylogenetic analysis revealed three major lineages (lineages A, B and C), which may represent A. fasciatus, A. wenchowensis and A. kreyenbergii, respectively. Our results revealed that A. fasciatus and A. wenchowensis diverged from A. kreyenbergii in the Zhejiang region. The uplift of the Wuyi Mountains and the Xianxia Mountains served as an important geographic barrier in the diversification of the three Acrossocheilus species. The most recent common ancestors (TMRCAs) of the three lineages and lineages A + B were dated to 1.859 and 1.614 myr, respectively. Our results indicate that the effective population sizes of A. fasciatus and A. wenchowensis remained constant from the past to the present, as supported by ABC analysis. For conservation and protection, a strategy is required because of their genetic uniqueness, and we suggest that the two regions divided by the Xianxia Mountains be regarded as different management units (Mus), conforming to the major zoological regions of the Zhejiang region.