To guide the breeding of (CA), one hybrid F₂[)], two backcross F₂[(BC1-F₂) and (BC2-F₂)] and their parents were analyzed using twenty microsatellite markers. The mean number of alleles (, and BC2-F₂ were 4.50, 4.40, 4.75, 4.85, and 5.10, respectively. The mean values of observed heterozygosity () were 0.7683, 0.5550, 0.7967, 0.8317, and 0.6200, respectively. The mean values of expected heterozygosity () were 0.6671, 0.6308, 0.6995, 0.7240, and 0.6949, respectively, and the average polymorphism information contents (PIC) were 0.6046, 0.5717, 0.6406, 0.6676, and 0.6339, respectively. The mean Hardy-Weinberg indices of MA, MC-F₂, BC1-F₂ displayed a heterozygote excess, whereas the gynogenetic populations (CA and BC2-F₂) showed a heterozygote deficit. The unweighted pair-group method with arithmetic means (UPGMA) showed that the CA and BC2-F₂ populations first grouped together, and then MA and BC2-F₂ grouped together, and then they clustered with MC-F₂. According to the genetic identity analysis, F₂ hybrids and backcross F₂ groups showed maternal effects. Particularly, the microsatellite primers Me.Am._15, Me.Am._1, TTF01, and Mam25 identified five groups. Growth performances of the five groups were analyzed further, and BC1-F₂ groups were found to have the most significant growth advantage. Our results provide important information for the breeding, germplasm resources preservation, and germplasm identification of hybrid F₂ (M. amblephala♀×(M. mblycephala♀×]. To cultivate hybrid strains with fast growth characteristics without adversely affecting the genetic background of the same or similar fish species in natural waters, backcrossing is a feasible method. This paper found that backcrossing could not only improve the superiority of varieties and facilitate correct selection, but also accelerate the stability of the heritability of hybrid offspring, reduce the separation of offspring, and make the heritability of hybrid offspring similar to that of the parent. Therefore, the backcross method lays a genetic foundation for artificial gynogenetic transgenic fish.