Abstract:[Objective] To create new germpla of Pleurotus ostreatus by mutagenic treatments protoplast by 60Co-γ-rays.[Methods] The protoplasts solution was treated by three different radiation doses of 60Co-γ-rays, we analysed the strain lethality rate, and screened the mutant strains by antagonistic reaction and simple sequence repeat (SSR) identification. Finally, we screened a new and fine strain of Pleurotus ostreatus.[Results] The strain lethality rate was 0% under 0.9 KGy, 63.33% under 1.2 KGy, and 76.67% under 1.5 KGy. After the antagonistic reaction and SSR identification, we found 8 mutant strains that were change. The fruiting results showed that the yield and biological efficiency of 7 mutant strains were lower than the control, and the cap color had some change. Compared with the control the yield of 1.5-P4 decreased 41.92%, but the 1.2-P1 was the high-yield strain with yield of 437.95±12.22 g/bag, increased by 9.76% compared with the control.[Conclusion] We screened a new and fine strain of Pleurotus ostreatus by mutation breeding with 60Co-γ-rays, and the results provide reference to mutation breeding of edible and medicinal fungi.