We developed and characterized 36 polymorphic microsatellite markers for the oyster mushroom (Pleurotus ostreatus). In total, 169 alleles were identified with an average of 4.7 alleles per locus. Values for observed (HO) and expected (HE) heterozygosities ranged from 0.027 to 0.946 and from 0.027 to 0.810, respectively. Nineteen loci deviated from Hardy-Weinberg equilibrium. Significant (P<0.05) excess heterozygosity was observed at nine loci. Linkage disequilibrium (LD) was significant (P<0.05) between pairs of locus alleles. Cluster analysis revealed that five species of genus Pleurotus made a distinct group, and the individual cultivars were grouped into major five groups from G-1 to G-5. The diverse cultivars of P. ostreatus were discriminated and the other four species revealed a different section in the UPGMA tree. These microsatellite markers proved to be very useful tools for genetic studies, including assessment of the diversity and population structure of P. ostreatus.