Using the whole genome sequence of PM_ss5 downloaded from GenBank database, genome-wide simple sequence repeats(SSRs) of Pleurotus pulmonarius were mined and characterized. A total of 2348 SSR loci were detected in PM_ss5 genome, with a relative abundance of 59 SSRs per Mb. Among all SSR loci, the di-nucleotide repeats were the dominant(51.8%), followed by the tri-nucleotide repeats(27.7%). The identified SSR loci contained 141 base motifs, among which the dominant base motifs were GA/TC and CT/AG. The length of SSRs ranged from 10 to 156 bp, and 77.2% of the identified SSRs were 10 to 15 bp. The SSR patterns in P. pulmonarius were also compared with those of other four species of Pleurotus. The short nucleotide repeats were dominant SSRs in all five Pleurotus spp., with the di-nucleotide SSRs accounting for higher rate in P. pulmonarius. Then, 53 pairs of polymorphic SSR primers were designed and selected to analyze the genetic diversity of the 18 varieties of P. pulmonarius, which showed P. pulmonarius owned moderate genetic diversity, and the average numbers of effective alleles, Nei’s genetic diversity and Shannon index were 1.35, 0.23 and 0.38, respectively.