The genetic diversity and sequence variation of mtDNA D-loop in the population of vulnerable species Onychostoma lini from the Duliu River in Guizhou were studied for the first time on the basis of determination of about 470 bp sequences of mtDNA D-loop in 36 individuals by the methods of PCR and DNA sequencing. The results showed that the length of the determined sequence in 36 individuals varied from 469 to 475 bp,and the total percentage of bases A and T (68. 4% ) was more than that of bases G and C (31. 6% ) in the sequence. A total of 25 polymorphic loci,including 19 transitions and 6 transversions,were detected in the sequence of the population. The nucleotide diversity (π) of the 36 individuals was 0. 005 75,and the average number of nucleotide differences (K) of them was 2. 695. The 36 individuals belonged to 5 haplotypes according to the determined sequences. The haplotypic diversity ( Hd) was 0. 260,and the average genetic distance ( P) between the haplotypes of the population was 0. 026. The UPGMA phylogenetic tree of the 5 haplotypes comprised 2 branches. The data show that the population of O. lini in the Duliu River has abundant variation in mtDNA D-loop sequence and high genetic diversity,which may play an important role in the conservation and recovery of the species.