Partial sequences of the mitochondrial cytochrome oxidase unit Ⅱ (COⅡ) gene of 23 species of Cyprinidae amplified by Polymerase Chain Reaction (PCR), and other two species of Cyprinidae from GenBank were treated by Clustal X and analyzed by PAUP 4.0，while selected outgroup was Crossostoma lacustre. 600 bp gene fragment from these species were obtained, in which 250 variable sites were identified, without insertions or deletions. The two types of molecular phylogenetic trees based on the sequences of 25 species with neighborjoining method(NJ) and maximum likelihood method(ML), respectively. The genetic distances among 25 species of Cyprinidae, measured by the kimura2 parameter model, were from 0.018 5 to 0.237 5. The least genetic distance was 0.018 5 between Hemiculter leucisculus and Toxabramis swinhonis, whereas the largest was 0.237 5 between Paracanthobrama guichenoti and Acheilognathus macropterus. Leuciscinae, Culterinae, Cyprininae, Gobioninae, and Acheilognathinae did not form monphyletic group respectively. Leuciscinae was divided into southern group and east Asian group. The southern group of Leuciscinae was monphyletic, while east Asian group of Leuciscinae, Culterinae, Xenocyprinae and Hypophthalmichthyinae formed a monphyletic group. Tinca tinca, Leuciscus leuciscus baicalensis, Abramis bramaorientalis and Rutilus rutilus lacustris formed a monphyletic group, which supported that tinca tinca should be kept in leuciscinae.