Abstract: To clone the sequence of the mitochondrial cytochrome b (Cytb) gene of Silurus lanzhouensis, we designed a specific pair of primers from the mitochondrial genome of S. glanis. The 1138 bp S. lanzhouensis Cytb gene was obtained after PCR. Then we compared the homology of the mitochondrial Cytb gene between S. lanzhouensis and 13 other species. The results showed that these species have high homology in Cytb gene. The homology value of the nucleotides was between 61.38% and 91.12%, and the value of the amino acids was between 76.62% and 95.52%. We also analyzed the nucleotides substitution of the mitochondrial Cytb gene among S.lanzhouensis, S.glanis, S.meridionalis, S.asotus, and S.cochinchinensis, and found that the lowest frequency of nucleotides substitution occurred between S.lanzhouensis and S.asotus with the value of 8.87%, and TS/TV was 3.21. The highest frequency was between S.lanzhouensis and S. cochinchinensis with the value of 14.41%, and TS/TV was 1.83. Qing-Rong Wang, et al. previously reported the sequence of the mitochondrial Cytb gene of S.lanzhouensis. Here we conducted the sequence difference analysis between our results and theirs. The results showed that the frequency of nucleotides substitution was 11.16%, there were 127 variable sites, TS/TV was 4.08, genetic distance was 0.1230. The phylogenetic tree of S.lanzhouensis and 13 other species constructed by NJ method was consistent with the traditional taxonomic results.