The mitochondrial COI gene was PCR amplified and sequenced from 17 samples obtained from three populations of Macrobrachium rosenbergii (F1 of the Burma wildtype population, Jiangsu cultured population and F2 of the Guangxi breeding population). A 498-bp long partial gene segment was acquired and used to study the genetic diversity among the three populations. Results indicated that the COI gene locus was relatively more polymorphic in the F1 of Burma wildtype population, while the polymorphism in Jiangsu cultured population and Guangxi breeding population were very poor. A total of 10 polymorphic sites and 5 haplotypes were found in the sequences of the 17 samples. The average nucleotide divergence in the three populations was 0.88%0.07% and 0 respectively. The UPGMA phylogenetic tree suggested that F2 of the Guangxi breeding population and Jiangsu cultured population were closest genetically, and their haplotypes could be gathered together to a genetic branch while F1 of the Burma wildtype population diverged and could form another relatively independent branch. For these distinct nucleotide differences, COI gene could be suitable as a genetic marker for distinguishing these two branches of the Macrobrachium rosenbergii population.