Oriental river prawn (Macrobrachium nipponense) is one of the most important freshwater prawns for aquaculture in China. However, M. nipponense farming recently declined due to genetic retrogression. Its genetic diversity of different populations in the Yangtze River with microsatellite markers was investgated in order to give support to conservation and utilization of wild resources of M. nipponense. Twenty polymorphic microsatellite markers were applied to investigate the genetic diversity of 6 M. nipponense populations in the Yangtze River. The sampling sites included Chongqing, Wanzhou, Yichang, Wuhan, Jiujiang and Jiangyin. The average number of alleles (A) and effective numbers of alleles (N_e) in 6 populations were 5.25 and 3.462 2 respectively. The mean PIC value of 20 microsatellite loci was 0.589 4. Expected heterozygosity (H_e) of 6 populations was as follows: Jiangyin (0.630 8), Jiujiang (0.609 6), Yichang (0.594 5), Wuhan (0.593 4), Wanzhou (0.584 4) and Chongqing (0.582 1), and their mean value was 0.629 6. The analysis of molecular variance (AMOVA) indicated that almost majority of the variance in the M. nipponense was within populations (93.08%) ，and 6.92％was among populations. The F_ST values between populations were 0.025 3-0.083 8(P<0.05), which showed the genetic divergence between populations in the Yangtze River was intermediate but lower than that in the lakes. It was probably because running water promoted the exchanges between populations. HardyWeinberg equilibrium analysis indicated that deficiency of heterozygote existed in all of 6 populations, which was probably because of rare allele deficiency or null alleles. The genetic distances among populations ranged from 0.062 0 to 0.180 9. The UPGMA tree showed that Jiangyin stock formed an independent clade and resident 5 populations formed another one in which Jiujiang and Wuhan populations clustered at first.