The present study examined the genetic structure and variation among different generations of Manila clam Ruditapes philippinarum obtained through selective breeding by using 12 microsatellite markers. Four consecutive generations (F₁, F₂, F₃, and F₄) were selected with 10% selection intensity from a wild population from Shihe, Dalian (Liaoning Province, China). Thirty six individuals were sampled from each generation. Among the 12 microsatellite loci, 121 alleles were detected. The number of alleles on each locus varied from 2 to 6 and the allele size ranged from 101 to 273 bp. Each generation had average effective alleles of 3.75-4.58 and average observed heterozygosity of 0.3391-0.3860. Based on P values of genotypes, all four generations deviated from the Hardy-Weinberg equilibrium with varying degrees. Of the 48 generation × locus combinations (4 generations × 12 loci), 27 of which were not in an equilibrium state. Except the locus Asari24, all polymorphic loci had positive inbreeding coefficients (F_is ), indicating that high degree of inbreeding existed in all four generations. The inter-generation fixation indices (F_st) varied from 0.0338 to 0.2136 and there was significant genetic differentiation among the 12 polymorphic loci. The F-statistics showed that among all generations two loci had weak genetic differentiation, eight loci had moderate, and the remaining two loci had high genetic differentiation. Pairwise F_st values ranged from 0.05 to 0.15, which also indicated that the clam had moderate genetic differentiation among the generations. Based on the F_is values, two out of four generations exhibited heterozygote excess, but all the generations in sequence showed some degree of heterozygote deficiency. The Nei's genetic similarity decreased (0.8203→0.8107→0.8031) and genetic distance increased (0.1918→0.2099→0.2129) from one generation to the next through selective breeding. The genetic similarity coefficients among the different generations ranged from 0.7873 to 0.8685, and genetic distance varied from 0.141 to 0.2391. These results demonstrated that there was moderate genetic differentiation among the four generations. In the present study, overall average polymorphism information content (PIC) of 0.5055 was observed, indicating that there is great potential for future selections of Manila clam through selective breeding.