Molecular markers were crucial role in understanding and using genomic information for marker-assisted breeding, mapping genes of interest and cloning genes. Among the molecular markers, Simple Sequence Repeat (SSR) has became increasingly important marker due to their co-dominant and high polymorphic nature and abundant distribution throughout the genome. Simple sequence repeats (SSR), also called “microsatellites” consist of tandemly repeated short DNA sequence motifs and have applied in various marker-based studies. SSRs were isolated and characterized from “Heuktari” and “Miso”, which are one of the major oyster mushroom cultivars in Korea by the genome sequencing and bioinformatic analysis. The genome sizes of “Heuktari” were 40.8 Mb and “Miso” were estimated to be 40.3 Mb, which were larger than the other P.ostreatus species (PC9 and PC10) and less than P.eryngii (KNR2312 P5). A total of 949 and 968 SSRs were found in the “Heuktari” and “Miso” genomes, respectively. A comparative analysis of five mushrooms including P.ostreatus var. florida (PC9 and PC15), P.eryngii, revealed that SSR numbers from “Heuktari” and “Miso” were the highest among them. Studied all mushrooms showed similar pattern of SSR distributions. Tri-, hexa- and octanucleotide motifs accounted for the top three fractions of all SSRs. This study are useful in understanding the P.ostreatus.[Supported by a grant from the IPET (213007-05-1-SBI30), MIFAFF, Republic of Korea.]