After high-throughput sequencing of tissue samples from Malaysian wild (MW) giant freshwater prawn (Macrobrachium rosenbergii), twenty-five identified single nucleotide polymorphisms (SNPs) were selected to be the markers for genetic diversity analysis. Additionally, SNP analysis was performed to investigate the genetic diversity of five prawn groups: Shanghai cultured group (SH), Zhejiang cultured group (ZJ), Malaysia cultured group (MF), Malaysia wild group (MW) and breeding-growth strain group (BG). The expected heterozygos (H_e) and observed heterozygosity (H_o) of the genetic diversity analysis were 0.28-0.37 and 0.18-0.30 respectively, and the average polymorphic information content values of five prawn groups were 0.29, 0.24, 0.21, 0.33 and 0.26, respectively. The results of the present study indicated that the polymorphism of five prawn groups was at an intermediate level. The minimal value of genetic similarity coefficient as well as maximal genetic distance was detected between MF and BG groups, on the contrary, the SH and ZJ groups showed the highest similarity coefficient and least genetic distance. Furthermore, the highest value of genetic differentiation coefficient and least gene flow were simultaneously observed between MF and MW groups. The least differentiation coefficient and the highest gene flow were detected between SH and ZJ groups. AMOVA estimated the variation of 26.33% between groups. The inbreeding coefficient was between 0.151 and 0.342. The average inbreeding coefficient of the three cultured populations was higher than that of the MW group. At 9 out of the 25 SNP loci in five groups, the result of the genotype analysis of the BG group is contrary to the other four groups. What’s more, at 14 out of 25 SNPs, the number of heterozygous individuals is generally decreased in three cultured groups compared with the MW group. The findings of the present study provide a novel insight into the molecular basis of genetic breeding of M. rosenbergii through the specific SNPs.