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Nucleotide sequences of the cytochrome b gene and D-loop control region of mitochondrial DNA (mtDNA) were used to assess the population genetic structure of Onychostoma lepturum, a Cyprinidae fish in Hainan Island.Intotal, 63 specimens are collected from four populations.The population analyses indicated significant genetic structure (FST=0.749).All 31 mtDNA haplotypes were identified as four lineages, and these lineages were almost allopatric distribution.