An enrichment protocol was used to isolate and characterize tri-nucleotide microsatellite markers in Crassostrea hongkongensis, an economically important and extensively cultured oyster species along the southern coastal areas of China. Thirty tri-nucleotide microsatellite loci were assessed in 32 individuals from a wild population. The number of alleles ranged from 2 to 12, averaging 7.63 alleles per locus. The observed and expected heterozygosity values ranged from 0.031 to 0.844 (averaging 0.579) and 0.062 to 0.881 (averaging 0.665), respectively. Six loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction. BLAST analysis showed that the tri-nucleotide microsatellite markers randomly developed from the genome of C.hongkongensis have a higher correlation with functional genes than di-nucleotide markers. Cross-amplification in C. ariakensis and C. gigas was performed and 21 loci showed reasonable polymorphism between C. hongkongensis and C. ariakensis, while 15 loci showed reasonable polymorphism between C. hongkongensis and C. gigas. The tri-nucleotide microsatellite markers identified in this study have greater potential for future use in gene mapping, molecular-marker assisted selection and comparative linkage mapping than previously identified di-nucleotide markers. Additionally, we found two microsatellite loci that can effectively identify the purebred and hybrid offspring of these three oyster species.