Abstract: Genetic diversity and genetic structure of five populations of Macrobrachium rosenbergii from Thailand (CP), Myanmar (MN), Bangladesh (BD) and China (MP and DP) were analyzed with 16 polymorphic microsatellite markers. The results showed that 16 microsatellite sites had high polymorphism, with average allele number (N_a), expected heterozygosity (H_e), Shannon information index (I) and polymorphic information content (PIC) at 17.563, 0.8316, 2.1662 and 0.733, respectively. The expected heterozygosity (H_e) of five populations was between 0.7025 and 0.8594, and the PIC was between 0.6538 and 0.8048, indicating that all populations had high genetic diversity. The level of genetic diversity was CP>MP>BD>MN>DP. The value of genetic differentiation index (F_st) was between 0.03430 and 0.17333, indicating different degrees of genetic differentiation among all populations. The F_st values of these 16 microsatellite sites were all greater than 0.05, with an average of 0.0977, which was consistent with the genetic differentiation among the populations. Molecular variation analysis (AMOVA) revealed that about 6.22% genetic variation was derived from inter-populations, 40.72% variation was derived from intra-populations and 53.07% variation was derived from intra-individuals. The UPGMA phylogenetic tree based on Nei’s genetic distance showed that the MN population clustered with the MP population first, then with the DP population, then with the BD population, and finally with CP population, indicating that the Chinese populations were far from the Thailand populations. The genetic structure simulation analysis suggested that there were five logic populations in all samples. Except for the mixed genetic structure of individuals in the MP population, the genetic structure of individuals in the remaining populations was relatively independent. This study provides reference data for the development and utilization of the germplasm resources and the breeding of superior species of Macrobrachium rosenbergii.