Six microsatellite loci were identified, characterized and evaluated from the ‘western’ form of wild Macrobrachium rosenbergii. The microsatellite regions identified were (GA/CT) 38 , (CA/GT) 13 , (GA/CT) 44 , (GA/CT) 26 , (GA/CT) 35 and (GA/CT) 33 repeats respectively. To study polymorphism in the microsatellites, PCR primers were designed for the flanking region and further tested on genomic DNA from wild M. rosenbergii samples collected from 2 different regions. The total number of alleles for the two different giant freshwater prawn populations was found to be 18 and 19 respectively. The average PIC value for these six microsatellite markers was found to be 0.70 indicating their very high level of polymorphism. The direct count heterozygosity values for these marker loci during multi-population analysis range from 0.4093 to 0.7035. F st values showed significant differentiation between the tested two populations. The populations were found to be in Hardy–Weinberg equilibrium, but one microsatellite loci was found to be deviated from HWE in both populations. One microsatellite loci MRMA27 in one population exhibited the presence for a null allele. The results indicate that these six microsatellite loci are highly polymorphic and can be used as genetic markers for M. rosenbergii populations.