Hypsizigus marmoreus is commercially the most important edible mushroom in Japan. This mushroom is usually cultivatedfor a longer period (about 85~120 days) than other mushroom. In order to develop a new cultivar that has a shortened cultivationperiod, the genome analysis of this strain has been considered. This study aims to obtain parental monokaryotic strains reproducing‘Haemi’ cultivar in Hypsizigus marmoreus for reference genome sequencing. The mycelia were cultured in MCM and MYG media forvarious incubation periods. Homogenized mycelia were treated with commercial cell wall degrading enzymes to maximize protoplastsproduction yield from Hypsizigus marmoreus. The greatest number of protoplasts was obtained from mycelia cultured in MCM mediafor 3 days using Novozyme enzyme. The isolated protoplasts were grown in regeneration agar media after two weeks. Regeneratedcolonies were picked and moved on separated dishes for microscopic observation. Neohaplonts regenerated from dikayotic strainswere identified by the absence of clamp connections. We confirmed that one of monokaryotic strains is a parental strain by crossingwith an original compatible strain of ‘Haemi’ cultivar. This parental strain will be used for reference genome sequence analysis.