Ruditapes philippinarum has a high growth rate, short culture cycle, and is highly adaptable. Because of these traits, it is one of China's four major cultured shellfishes, and one of the world's major cultured shellfishes. Microsatellites known as simple sequence repeats are widely used to assess genetic diversity in farmed aquatic species populations, construct molecular genetic maps, and carry out gynogenesis, gene mapping, gene cloning, and paternity tests. These molecular markers have high stability and polymorphism, are site-specific and easily detected, and exhibit codominant inheritance and transferability of SSR primers. At present, the sustainable culture of Ruditapes philippinarum is threatened by having a single breeding method and difficulties with disease prevention, control, and treatment. We developed a series of microsatellite markers using a transcriptome-based platform to provide a foundation for genetic research in Ruditapes philippinarum. These markers may also be used for Ruditapes marker-assisted breeding. Genetic diversity measures the degree of variability of biological genetic information. DNA is the primary carrier of genetic information, so the diversity of DNA directly reflects the degree of genetic variation. The genetic diversity of a population can be represented by the number of alleles, heterozygosity scores, and polymorphism information content (PIC). We sequenced a large number of ESTs and screened 145 potential microsatellites of trinucleotide repeats using MISA software. We successfully obtained clear, reproducible bands for 58 microsatellite loci. These were amplified in 32 wild clam individuals sampled from Zhuanghe, Dalian, Liaoning. A single allele was detected at 18 loci and another 40 were polymorphic (number of alleles per locus ranged from 2 to 6, with an average of 3.4250±0.9718). The observed and expected heterozygosity was 0.000-1.000 (0.2727±0.2272) and 0.0615-0.7996 (0.4739±0.1902), respectively. The average of the Nei index was 0.4664±0.1872. The polymorphism information content (PIC) of all loci ranged from 0.0586 to 0.7529 (0.4148±0.1707). Among these, 16 loci had a PIC of > 0.5, so were classified as highly polymorphic. An additional 15 loci were moderately polymorphic with PICs ranging from 0.25 to 0.5. The PIC of 9 loci was < 0.25, meaning that these were classified as low polymorphic loci. Using a test of the Hardy-Weinberg principle (χ² test) and sequential Bonferroni calibration, all except 10 loci had deviated equilibrium. Eight loci had a core sequence of TTG, 6 had a core sequence of TGG, and 5 each had a core sequence of TGT or ATC. These four core sequences accounted for 41.38% of the 58 SSR screened loci. Based on this, we infer that TTG, TGG, TGT, and ATC are relatively abundant copy categories of the trinucleotide repeat sequences. Our results provide a reference for subsequent repetitive sequence screening and further understanding the characteristics of the Ruditapes genome. Microsatellite marker development by transcriptome sequencing proved to be efficient and feasible in R. philippinarum . Further in-depth analysis based on transcriptome analysis will likely yield more microsatellite sites which are associated with functional genes, thereby providing more molecular markers for Ruditapes artificial marker-assisted breeding. These polymorphic markers may also be used in future studies of population genetics, linkage mapping and assisted breeding in R. philippinarum.