To guide the subsequent breeding of hypoxia-tolerant population of Megalobrama amblycephala; genetic structures of hypoxia-tolerant (TN), meiotic gynogenesis (TNM), mitotic gynogenesis populations (TNDH) of M. amblycephala were analyzed using twenty microsatellite markers in this study. The mean value of number of alleles (N_a) of M. amblycephala TN, TNM, TNDH and the control TPJ1 was 3.90, 3.55, 3.45 and 2.56, respectively. The mean value of observed heterozygosity (H_o) was 0.7853, 0.3934, 0.2768 and 0.8075, respectively. The mean value of expected heterozygosity (H_e) was 0.6491, 0.5563, 0.4870 and 0.6855, respectively. And the average value of polymorphism information content (PIC) was 0.5695, 0.4796, 0.4181 and 0.6105, respectively. It demonstrated that the genetic diversity of TPJ1 was the highest, and TN population had lower genetic diversity, but there is no significant difference and there was the high genetic heterozygosity. The genetic diversity of TNDH population was significantly lower than TNM population, and the genetic diversity of the gynogenetic (TNM and TNDH) was significantly lower than TPJ1 and TN populations. The mean Hardy-Weinberg index of the TPJ1 and TN had heterozygote excess and the gynogenetic populations (TNM and TNDH) showed heterozygote deficit. Using unweighted pair-group method with arithmetic means method (UPGMA) based on their genetic distances, TPJ1 population and TN population were grouped in one cluster, while TNM population and TNDH population were classified into another cluster, indicating a genetic differentiation between the two clusters. Our results show that the hypoxia-tolerant group in M. amblycephala (TN) in gynogenesis can accelerate homozygosity of genetic material and will have further purification of M. amblycephala hypoxia-tolerant genes.