[Objective] Simple sequence repeat (SSR) molecule markers were used to separate and identify protoplast monokaryons, sporulated monokaryons and their hybrid progenies of two Xianggu (Lentinula edodes) strains. [Methods] SSR primers developed from the whole genome sequence of Xianggu were used to identify the genotypes of different monokaryons and hybrids, and then to separate them according different genotype information. [Results] Employing Lefp-55 SSR markers, we obtained both protoplast monokaryons of strain “L808” without matching crossing of monokaryons, and we found the segregation ratio of both protoplast monokaryons was high to 191:1. This result was confirmed by other SSR markers, random amplified polymorphismic DNA (RAPD) markers and tranditional method. In the identification of sporulated monokaryons and their hybrid progenies, cooperation of several SSR markers was able to identify numerous hybrids, and it was useful in genetic and breeding research. [Conclusion] Protoplast monokaryons separation would be fast and accurate by using SSR markers, which also can be used in identification of sporulated monokaryons and their hybrid progenies of Xianggu in relevant genetic and breeding research.