Abstract: Mitochondrial COI gene can provide important information for species identification and phylogenetic studies within family.【Objective】To explore the feasibility of COI gene as a molecular marker for species identification of Macrophthalmidae by comparing the differences in mitochondrial COI gene sequences of Macrophthalmidae species. At the same time, comprehensive phylogenetic tree of Macrophthalmidae was constructed based on COI gene sequence, which provided a theoretical basis for the phylogenetic study of Macrophthalmidae.【Method】In this study, the complete COI sequence of 3 Macrophthalmus species, Macrophthalmus latreillei, M. japonicas and M. pacificus were determined and other 18 partial COI sequences of Macrophthalmidae species were downloaded from GenBank database. Multiple sequence alignment analysis was performed using MatGAT 2.02, and phylogenetic trees were constructed using the maximum likelihood(ML) and maximum parsimony(MP) methods in MEGA X with Portunus trituberculatus and P. sanguinolentus as outgroups.【Result】COI gene full-length sequence of M. latreillei, M. japonicas and M. pacificus was 1534 bp, encoding 511 amino acid residue, and ATG was the start codon and T was the incomplete stop codon. Sequences for multiple sequence alignment was 657 bp, encoding 219 amino acids continuously, with 28.9%-35.9% T, 18.9%-27.2% C, 25.3%-29.7% A, and 16.6%-19.0% G;respectively. Sequence alignment of nucleotide and amino acid sequences revealed 267 and 20 variable sites, with most of the variation sites appeared in the third codon, and the second codon was very conservative, which showed the degeneracy of genetic code. The genetic distance, sequence similarity and phylogenetic analysis showed M. japonicas and M. pacificus had the closest relationships with M. banzai and M. tomentosus; respectively. However, further studies were needed to evaluate the evolutionary status of M. latreillei and the monophyletic origin of Macrophthalmus.【Conclusion】The mitochondrial COI gene sequences of 21 Macrophthalmidae species are different from each other, which can be considered as a molecular marker for species identification. However, it is still insufficient to analyze the phylogenetic relationships between certain species because it contains too few effective genetic sites and the genetic relationship between some species is less reliable, so subsequent studies should be based on more gene sequences and more species to solve this problem.