Oriental river prawn Macrobrachium nipponense is the most important commercial species in China. Mitochondrial cytochrome oxidase subunit I (COI) gene was used to analyze the genetic diversity and genetic structure. A fragment of the COI gene was amplified and sequenced from 100 wild M.nipponense individuals obtained from 5 lakes of China: Poyang Lake (PY), Dongting Lake (DT), Tai Lake (TH), Chao Lake(CH) and Hongze Lake(HZ). Forty nine nucleotide variable positions were detected in analyzed 607 bp COI sequences, and 35 haplotypes were defined among five populations, but only seven shared haplotypes was observed. Each population had good haplotypic diversity and nucleotide diversity, and among them PY shows the best genetic diversity. The fixation indices (Fst) analyzed by AMOVA was total to 0.31873 (P<0.05), which showed significant genetic fixation among the five populations. The Kimura 2-paramter genetic distance calculated by the MEGA version 3.1 between the population of PY and CH was highest up to 0.0191, while that between the population of HZ and CH was the lowest to 0.0051. The neighbor-joining trees and UPGMA trees from haplotypes and populations of M.nipponense indicated that, the population of PY and DT might have originated from one clade, while population of TH, HZ and CH might have originated from another clade.