In order to verify the feasibility of DNA barcoding in the identification of Macrobrachium species, this study analyzed the sequence of COⅠ gene fragments of 21 species of Macrobrachium. The results showed that the COⅠ gene composition of the Macrobrachium was significantly biased, with higher AT content(57.74%) and lower GC content(42.26%). The content of GC at each codon site from high to low is the first codon(49.24%), the second codon(40.72%), and the third codon(36.82%). Based on the Kimura 2-Parameter model, the interspecific and intraspecific genetic distances of 21 species of Macrobrachium were 0.753 and 0.005, respectively. The average genetic distance between species was 150.6 times of the intraspecific genetic distance, which was consistent with the minimum effective interspecies genetic distance for identifying different species is 0.02 and the genetic distance between species is greater than or equal to 10 times the genetic distance within the species recommended by Hebert. In the molecular phylogenetic tree, the topological structure of the NJ tree and the ML tree are basically the same, and the Asian and American populations form two sister branches with 100% and 82% confidence, respectively, and the same species of Macrobrachium are clustered in the same branch with high confidence. The results show that the mitochondrial COⅠ gene can accurately identify the species of Macrobrachium.