Abstract: 【Objective】 To comparatively analyze the sequence differences of mitochondrial COI gene in 20 Potamidae species. The phylogenetic relationship of Potamidae species was constructed based on the COI gene sequences to clarify the evolution of Potamidae, to explore the applicability of COI gene as molecular marker in species identification of Potamidae, and to provide theoretical basis for species identification and phylogenetic studies of Potamidae.【Method】 The complete mitochondrial COI gene sequences of two representative species of Longpotamon were determined. Combined with the complete mitochondrial COI gene sequences of 18 species of Longpotamon published in GenBank, MEGA X was used to calculate the base composition, conserved loci and genetic distance, and MatGAT 2.02 was used to analyze the multi-sequence similarity. And PhyloSuite constructed Bayesian trees(BI) and maximum likelihood trees(ML) to explore the internal phylogenetic relationships of species in Potamidae.【Result】The COI gene sequence of 20 species of Potamidae was 1534-1539 bp, encoding 511-512 amino acid residues, and all species started with ATG as the codon. Base contents were slightly different, which were 35.9%-40.7%(T), 16.4%-20.2%(C), 26.8%-28.9%(A) and 14.7%-17.2%(G), and it was obvious AT bias. The nucleotide sequences of COI gene and its derived amino acid sequences showed 577 and 98 variation sites, respectively, indicating the existence of codon degeneracy. The genetic distance, sequence similarity and phylogenetic evolution of mitochondrial COI genes of 20 species of Potamidae showed that Longpotamon changanense was the closest relative to Longpotamon sp. Although phylogenetic trees constructed by different methods and based on different data sets differed in topology, all tree types showed that L. parvum of Longpotamon was not clustered with other species in the genus. Sinolapotamon, Potamiscus and Tenuilapotamon species were also scattered in the phylogenetic tree, suggesting that these species were non-monophyletic groups in taxonomic identification and needed further study to confirm.【Conclusion】 The mitochondrial COI gene sequences of 20 Potamidae species have average genetic distance of 0.173, and they all have specific sites differing from each other. Therefore, mitochondrial COI gene sequences can be considered as a molecular marker for classification and identification of Potamidae.