Abstract: ObjectiveGenetic diversity of cytochrome b (Cyt b) gene in populations of Silurus asotus and Hemibagrus guttatus in Duliujiang River, Guizhou was studied, in order to supply theoretical basis for resource protection and ex-ploitation of commercial siluriformes fishes in Duliujiang River. MethodCyt b genes in populations of S. asotus and H. guttatus in Duliujiang River were amplified using PCR. After bidirectional sequencing and splicing , sequence variation and genetic diversity of Cyt b gene were analyzed using related online softwares. ResultCyt b gene sequences of the two pop-ulations comprised of 1122 bp. Forty-six variable sites were detected in S. asotus population while fifteen in H. guttatus population, and they were identified as eleven haplotypes (NHap1-NHap11) and ten haplotypes (BHap1-BHap10) respec-tively. Haplotype diversity and nucleotide diversity of S. asotus population were 0.646 and 0.00899, whereas those of H. guttatus population were 0.610 and 0.00101. Cyt b gene sequences in the two populations encoded 374 amino acids, in-cluding 20 kinds of amino acids. The content of leucine was the highest (17.65%in S. asotus population and 16.58%in H. guttatus population) and that of cysteine the lowest (0.80% in S. asotus population and 0.78% in H. guttatus population). Occurrence frequencies of four bases at the first site in the two populations were almost the same , but the occurrence fre-quencies at the second and third sites in the two populations were significantly different. Variation frequencies of Cyt b gene codons at the third site in S. asotus population and H. guttatus population was significantly higher than those at the first and the second sites. But the variation at the third site was synonymous mutation in most cases. ConclusionS. asotus population is stable in quantity and has high genetic diversity; whereas H. guttatus population has low genetic diversity and probably experienced bottleneck effect and expansion of population. Therefore it is necessary to protect and recover H. guttatus population in Duliujiang River.