This study was performed to develop new Hypsizygus marmoreus cultivars that have enhanced functional materials and improved physiological characteristics with mutagenesis by gamma ray irradiation. Protoplasts of H. marmoreus brown strain HYM-056 were irradiated by gamma ray for mutagenesis, and then 2,000 clones of mutants were randomly selected and the fruiting bodies were induced by bottle culture. Among them, 157 isolates with fast-growing, heavy and many fruiting body-producing were selected. The isolates were cultured in plastic bottle containing rice bran, barley hulls and fir sawdust to form the fruiting bodies. About 100 days after inoculation, characteristic of fruiting bodies were investigated. The isolates were divided into 6 groups based on color, shape and size of pileus, and length, diameter, number and weight of stipe. In addition, the genetic variation of the isolates was analyzed by URP-PCR fingerprinting.