Abstract: To learn the genetic background of the giant freshwater prawn (Macrobrachium rosenbergii), four different breeding populations, including Shufeng nucleus breeding population selected for three generations (SF), introduced Zhengda population (ZD), hybrid population between the introduced Zhengda population and Shufeng population (ZDS), and hybrid population between the progeny of the introduced Zhengda population and Shufeng population (ZD2S), were investigated by using both microsatellite loci and mitochondrial COⅠand 12S rRNA genes as molecular markers. The microsatellite data of 150 individuals revealed that all the seven loci had high diversity with PIC>0.5. The average number of alleles (N_a), expected heterozygosity (H_e) and polymorphism information content (PIC) were 19.43, 0.8980 and 0.8867, respectively. Among seven loci of four populations, the average H_e (0.874) and PIC (0.854) of the SF were the highest, followed by H_e (0.863) and PIC (0.834) of the ZD2S, and the lowest H_e (0.798) and PIC (0.761) of the ZDS. The pairwised genetic differentiation indices (F_ST) among the four populations was 0.04166—0.10438, suggesting low to moderate genetic differentiations. The F_ST between ZD and ZDS was the highest with a value of 0.10438. Based on the concatenated mitochondrial COⅠ and 12S rRNA gene sequences, 149 individuals contained 27 haplotypes, with the average haplotype diversity (H_d) and nucleotide diversity (π) of 0.846 and 0.00313, respectively. Among four populations, the H_d of the ZD2S was the highest, followed by the SF, and ZDS was the lowest. For the π values, the SF was the highest, followed by ZD2S, and ZD was the lowest, which was generally consistent with the genetic diversity results analyzed based on microsatellite data. However, the genetic differentiation among four populations based on mitochondrial genes was low with F_ST values of –0.02226—0.07310, which is much lower than those based on microsatellite. These results indicated that both SF and ZD2S maintain high genetic diversity and have the potential for further selective breeding of Macrobrachium rosenbergii.