To ascertain that genetic differences exist among different geographical populations of Manila clam Ruditapes philippinarum, 13 polymorphic microsatellite loci were isolated and characterized for three wild populations including the Dalian, Putian, and Qingdao populations. All samples were collected in March 2010. Among the three populations studied, 154 alleles were detected (there were 54, 51, and 49 alleles for the Dalian, Putian, and Qingdao populations, respectively). The number of alleles per locus ranged 2-7 and there were 1.2331-5.4568 effective alleles with an average of 2.7657. The observed heterozygosites for the Dalian, Putian and Qingdao populations were 0.4387, 0.4194, 0.2383, and the expected heterozygosities were 0.6488, 0.6484, 0.5526, respectively. The polymorphism information content (PIC) ranged 0.1778-0.7922 with an average of 0.5475, where the Rp-02, Rp-03, Rp-05, Rp-06, Rp-07, Rp-09, Rp-10, Rp-11, Rp-12 loci were considered highly polymorphic markers (PIC>0.5), and the Rp-01, Rp-04, Rp-08, Rp-13 loci were moderate polymorphic markers (PIC>0.25 and < 0.5). Genetic diversities among populations were not significantly different (P > 0.05). The genetic distance between the Dalian and Putian populations was only 0.1108 and they had the highest genetic similarity, whereas the genetic distance between the Dalian and Qingdao populations (0.4323) was farthest indicating that they were least genetically similar. The genetic distance between the Putian and Qindao populations was intermediate. The genetic distances within a population were 0.5178±0.1328, 0.6592±0.3291, and 0.5480±0.2759 for the Dalian, Putian, and Qingdao populations, respectively. The genetic similarities within a population were 0.6930±0.2708, 0.5426±0.1572, and 0.5986±0.1496, respectively. The NJ dendrogram also showed that the Dalian and Putian populations were more closely related to each other than with the Qingdao population. Most loci among the three populations deviated from the Hardy-Weinberg equilibrium; only 33.4% of the loci did not. The multi-group test showed that among groups of multiple loci detected, only the Rp-10 locus maintained in an equilibrium state, whereas all other 12 loci did not. This indicates that these populations have relatively low gene stabilities and genotype frequencies. Based on the inbreeding coefficient (F_is) of each population, the Qingdao population had only one locus that exhibited heterozygote excess, whereas two loci in the Dalian population and three loci in the Putian population showed heterozygote excess. Therefore, heterozygote deficiency is common in all three populations. The inter-population fixation index (F_st) was < 0.05 for the Rp-07 and Rp-11 loci, indicating that some degree of differentiation exists among the three geographical populations. We observed an average PIC of > 0.5, indicating that the microsatellite loci were highly diversified. Information obtained from the present study should be useful for analysis of genetic diversities among geographical populations, future development of genetic linkage maps for different populations, and the culture and breeding of Manila clam.