Abstract: Freshwater prawns (Decapoda: Caridea: Macrobrachium) play an important role in domestic fishery resources. Culturing M. rosenbergii and M. nipponense brings great economic benefits, as the two species were widely farmed in China. M. qilianensis, a native species with natural distribution limited in Gansu province, was classified into genus Macrobrachium based on external morphological characters. In order to understand the molecular genetic differences among the three species of Macrobrachium, i.e., M. rosenbergii, M. nipponense, and M. qilianensis, we analyzed sequences of mitochondrial cytochrome oxidase subunit I (COI) of them. It would provide theoretical basis of exploiting and utilizing Macrobrachium resources rationally. A total of 30 individuals (10 individuals of each species) were collected from Gansu and Zhejiang province. Samples of M. qilianensis were wild, however, that of M. rosenbergii and M. nipponense were cultured. Their mitochondrial COI gene segment sequences were obtained by using the method of PCR amplification and sequencing. After alignment, 649 bp consensus sequences of COI were obtained. One hundred and sixty-nine variable sites were detected in all 30 individuals, accounting for 26.04% of total sequence. A total of seven haplotypes were also detected. Nucleotide diversity was 0.411% within M. rosenbergii, 0.092% within M. nipponense, and 0.031% within M. qilianensis. Genetic diversity of wild M. qilianensis was much lower than that of cultured M. rosenbergii and M. nipponense. Genetic distances between different haplotypes of the three prawns ranged from 19.87% to 23.84%. It suggested that the three species were valid species, because genetic distances among them were quite great. To further determine the taxonomic status of the three prawns in family Palaemonoidae, we downloaded the corresponding COI sequences of Palaemonoidae prawns from Genbank and analyzed the phylogenetic relationships of them. Phylogenetic tree (NJ) showed that M. nipponense, M. rosenbergii and other Macrobrachium species constituted one monophyletic group. However, M. qilianensis, Exopalaemon carinicauda, and Palaemon debilis formed the other clade. Thus, results of COI sequences did not support that M. qilianensis belonged to genus Macrobrachium. The taxonomic status of M. qilianensis should be reevaluated with more comprehensive evidences.