In order to study the genetic diversity and taxonomic status of Gymnocypris chilianensis on a molecular level, the mitochondrial DNA cytochrome b gene was sequenced for 74 individuals of G. chilianensis from two locations (Heihe River and Shule River) and 42 individuals of its affinis species Gymnocypris przewalskii. Analyses of genetic diversity and sequence differences were conducted for these samples, combined with the analysis of 30 homologous sequences of another affinis species Gymnocypris eckloni, which were downloaded from GenBank. The results showed that both the haplotype diversity (h = 0.9820) and nucleotide diversity (π= 0.0039) of the Shule River G. chilianensis were lower than the other populations, thus, the Shule River G. chilianensis should be prioritized for protection because of its lower genetic diversity level. The results of sequence analysis showed that the genetic distance between the Heihe River G. chilianensis population and the Shule River G. chilianensis population was 0.0064, and the genetic distance between these two populations and the G. przewalskii population was 0.0838 and 0.0810, respectively. The genetic distance between the two G. chilianensis populations and the G. eckloni population was 0.0805 and 0.0778, respectively. Analysis of sequence differences indicates that G. chilianensis is sufficiently diverged from G. przewalskii and G. eckloni to the extent that it has reached species level, thus, G. chilianensis can be considered an independent species of Gymnocypris.