Abstract: In order to protect germplasm resources of Gymnocypris przewalskii scientifically and effectively, it was especially important to investigate the population structure and genetic polymorphisms of Gymnocypris przewalskii. 754 bp nucleotide sequences of mtDNA D-loop region were obtained from 155 individuals of four populations (Lake Qinghai, Lake Keluke, Ganzi River and Cao Dalian) by PCR amplification. The sequences were analyzed by using the MEGA (version 5.05) and DnaSP (version 5.10.1) software. The results showed that 34 haplotypes were detected among 155 individuals. The haplotype diversity (Hd) and the nucleotide diversity (Pi) were (0.906±0.013) and (0.00556±0.00034) respectively. The haplotype diversity and nucleotide diversity of Lake Keluke population were (0.422±0.093) and (0.00272±0.00066), which were lower than those of three other populations. The low level of gene flow (Nm<0.69959) and high population differentiation values (Fst>0.26327) between Keluke population and other populations showed considerable genetic isolation, while the highest genetic differentiation was detected between Lake Keluke population and Ganzi River population (Fst=0.45854, P<0.01; Nm=0.29521). But no monophyletic group was observed clearly in the phylogenetic tree. This may be the geographic barriers among the drainages were formed relatively late. It could be concluded that Gymnocypris przewalskii has high genetic diversity. It was a certain degree of differentiation among Gymnocypris przewalskii in different geographical populations, especially Lake Keluke population. But the genetic diversity of Lake Keluke population was low, so we should make it a priority to protect.