In order to understand further the genetic resources of Macrobrachium nipponense in South Baiyangdian (SB), North Baiyangdian (NB) and Hengshui Lake (HS) of Hebei Province, we investigated the genetic structure of Oriental river prawn M. nipponense among three different wild populations from Baiyangdian and Hengshui Lake of Hebei Province using nucleotide sequences from control region (D-Loop) gene of mitochondria. The results revealed polymorphisns at 80 nucleotide sites from an approximately 935 bp fragment of this gene region sequenced, accounting for 8.56% of all sites, of which 77 were parsimony-informative sites, the contents of A, T, G and C of D-loop were 42.65%, 36.98%, 9.08% and 11.29%, respectively, and the content of A+T (79.63%) was significantly higher than that of G+C. The fixation indices (F_ST) analyzed by AMOVA of ARLEQUIN Version 3.11 was 0.2336, which indicated that the variation percentages between and within groups were 23.36% and 76.64%, respectively. NJ phylogenetic tree of 23 haplotypes constructed by Kimura-2-parameter model showed haplotypes did not cluster in accordance with the group of haploid type, but characterized by different groups with individual interlaced form complex cluster. The mismatch distribution analysis and neutrality test demonstrated that M. nipponense has not experenced a population expansion recently.