The species Onychostoma rara, belonging to Cyprinidae, Cypriniformes, is endemic to China and distributed only in the Yuanjiang River and the Xijiang River systems. The species has been listed as the endangered one now. To evaluate the present state of genetic diversity of Onychostoma rara, the sequence polymorphism of mtDNA D-loop of the species is studied for the first time on the basis of determination of the 478 bp sequences from 3′ end of mtDNA D-loop in the 30 individuals of the species collected from the Qingshui River in Guizhou in Octomber, 2007, by the methods of PCR, clone and DNA sequencing. A total of 25 variable sites (about 5.23% of the total nucleotides in the sequence), including 23 transitions and 2 transitions with transversion, are detected in mtDNA D-loop of the species. The 30 individuals belong to 18 haplotypes respectively according to the determined sequences. The nucleotide diversity (π) of the 30 individuals is 0.0107, and the average number of nucleotide diffences (K) of them is 5.092. The haplotypic diversity (H) is 0.940, and the average genetic distance (P) between the haplotypes of the species is 0.014. The NJ phylogenetic tree of the 18 haplotypes comprises 2 branches. The population of Onychostoma rara in the Qingshui River includes abundant polymorphism in mtDNA D-loop sequence and high genetic diversity. The protection of population of Onychostoma rara from the Qingshui River is very necessary for the conservation and recovery of the species.