Simple sequence repeats (SSRs) were isolated from major Pleurotus pulmonarius cultivars in Korea, namely ‘HS47’ (monokaryon, gamete of ‘Santari’), ‘GB19’ (monokaryon, gamete of ‘Santari’), ‘Hosan,’ ‘Yeoleumneutali1,’ ‘Sambok,’ ‘Gangsan,’ ‘Yaksan,’ ‘Jasan,’ ‘Hyangsan,’ and ‘Yeoleumneutali2,’ and characterized via HiSeq genome sequencing and bioinformatic analysis. The genome sizes of the monokaryons ‘HS47’ and ‘GB19’ were estimated to be 37.3 and 37.2 Mb, respectively, and those of the other dikaryotic cultivars ranged from 47.1 to 61.1 Mb. A total of 711 (smallest) and 1,106 (1.5 times the smallest) SSRs were found in the ‘HS47’ and ‘Gangsan’ genomes, respectively. Hexanucleotide and octanucleotide motifs accounted for the top two fractions of all SSRs. CGA/TCG, A/T, and CTC/GAG were the most frequently detected nucleotides in the SSRs. Most of the SSRs were 21~30 nucleotides long (hypervariable for application), accounting for 70% of all lengths of SSRs.