As assessment of genetic structure of population is the key point of aquatic animal breeding, the selection of sample size and loci number is the first step of correct evaluation of population genetic structure. Here, we solved the problem from an analysis of genetic diversity of three breeding groups of Macrobrachium rosenbergii at 60 polymorphic microsatellite loci. The results show that selection of different sample sizes and loci numbers both had a great effect on the genetic diversity index. The sample size was positively correlated with the average number of alleles and the average effective number of alleles, and the sample size was moderate and highly correlated with heterozygosity and Nei's genetic diversity index, respectively. Only when the sample size exceeds 30, they tend to be stable. When we select the loci, we found the different polymorphism information contents also directly affect the accuracy of the genetic parameters. When the loci number was 5–25, there was a significant difference in the genetic diversity index(P<0.05). When the loci number was more than 25, the genetic parameters changed little(P>0.05). So, we suggested that when microsatellite markers were used to estimate genetic diversity of population as far as possible to choose high polymorphism information content and we can't consider sample gender, meanwhile the minimum loci number and sample size are needed to exceed 25 and 30, respectively.