The Manila clam, Ruditapes philippinarum, which is widely distributed along the coasts of China, is an economically important marine bivalve species in China's aquaculture industry. The world production of this species was 3.6 million metric tons in 2010. China is the first largest country in the world in terms of production of the Manila clam, producing about 3.0 million metric tons annually, which accounts for about 90% of global production. This species has several pedigrees including White, Zebra, Liangdao Red and Marine Red distributing in the coastal areas in North China. Microsatellites or simple sequence repeats (SSRs) are tandemly repeated motifs of 1-6 genetic base pairs. Microsatellite markers are powerful molecular markers due to their high polymorphism, stability, and co-dominance, and they are used widely in studies of genetic diversity, parentage assignment, genetic linkage map construction, and trait-related marker screening. In this study, 20 microsatellite DNA markers were used to analyze the genetic diversity of 107 individuals of a Zebra F2 pedigree of R. philippinarum. Forty-one alleles were detected, and the number of alleles (Na) was 2-3 at each locus (average, 2.05). The effective number of alleles (Ne) was 1.71, and the DNA fragment length was 109-430 base pairs. The mean values of observed heterozygosity (Ho), expected heterozygosity (He), and polymorphism information content (PIC) were 0.504, 0.431, and 0.324, respectively. The probability value of the chi-square test showed that three loci significantly deviated from Mendelian segregation (P < 0.01), which suggested that these loci might link with the adaptive gene, and two loci (SSR11 and SSR164) may link with recessive homozygous lethal genes. The general linear model procedure in SPSS20.0 was used to analyze the correlation between the 20 microsatellites and growth-related traits of R. philippinarum (i.e., shell length, shell width, shell height, and body weight). Four loci were significantly related to the growth traits (P < 0.05): SSR135 and SSR164 were significantly related to shell width (P < 0.05), SSR9 was significantly related to shell height (P < 0.05), and SSR142 was significantly related to total weight (P < 0.05). Favorable genotypes for each growth trait were identified by a multiple comparison among the loci. Allele A for SSR9 had a significant impact on shell height (P < 0.05) and had the highest phenotype value; thus, it can be used as a molecular marker for selective breeding. SSR135 and SSR164 had a significant impact on shell width and total weight (P < 0.05), respectively, and allele B for SSR135 and allele A for SSR142 had a positive effect on growth-related traits. The following four genotypes of these loci had a favorable effect on growth-related traits: AA for SSR9, BB for SSR135, AA for SSR142, and AB for SSR164. The trait-related microsatellite loci identified in this study will be valuable for marker-assisted breeding of R. philippinarum.