To breed a Megalobrama amblycephala “Pujiang No.2” pure strain, M. amblycephala varieties “Pujiang No.2” were selected as breeding parents, using UV-inactivated carp sperm stimulation of M. amblycephala eggs. Cold shock was used to inhibit second polar body eduction for the heterogeneous gynogenesis (Meio-G1) and gynogenesis second- (Meio-G2) generation groups. The genetic characteristics of “Pujiang No.2” normal population, Meio-G1, and Meio-G2 populations of M amblycephala were analyzed using twenty pairs of microsatellite primers. The results showed that 129, 99, and 84 alleles were amplified from the normal population, Meio-G1, and Meio-G2, respectively. The average number of alleles (Na) was 4.30, 3.30, and 2.80, and the average number of effective alleles (Ne) was 3.23, 2.24, and 1.76 for the three groups, respectively. The average observed heterozygosity (Ho) was 0.8067, 0.4067, and 0.1733; the average homozygosity (H) was 0.2035, 0.6000, and 0.8263; and the average polymorphic information content (PIC) was 0.6198, 0.4701, and 0.3628, respectively. The results showed that the genetic diversity of Meio-G1 and Meio-G2 decreased compared to that of the normal population, and the genetic diversity of Meio-G2 was the least. The mean Hardy-Weinberg index of Meio-G2 and Meio-G1 showed a heterozygote deficit, as did the gynogenetic normal population. Using the unweighted pair-group method with arithmetic means method based on their genetic distances, the normal and Meio-G1 populations were grouped into one cluster, while the Meio-G2 population was classified into another cluster, indicating a genetic differentiation between the two clusters. The results showed that Meio-G2 was a good breeding material, with high purity and low genetic diversity, providing an important reference value for breeding of Megalobrama amblycephala.