Abstract: 【Objective】To analyze the genetic diversity of Stropharia rugosoannulata and obtain new strains through atmospheric and room temperature plasma mutagenesis technology, which could provide theoretical reference for selecting and breeding suitable local cultivars according to local conditions and the development and utilization of S. rugosoannulata. 【Method】Ten strains of S. rugosoannulata（numbered D1-D10） were activated for culture, colony diameter was measured, mycelia uniformity, density and other growth conditions were observed and recorded, and mycelia growth rate was calculated. PCR was used to detect 10 strains by Random amplification of polymorphic DNA（RAPD） molecular marker technology, and the genetic relationships among different strains were analyzed by RAPD genetic diversity cluster analysis. The strain with the fastest mycelium growth rate was selected as the original strain（CK）, and after mutagenesis treatment of its protoplasts, the mutagenic strain with obvious antagonism to CK was selected, the mycelium growth rate of different subculture mutagenic strains was determined, and the mutagenic strain and CK were detected by RAPD-PCR. 【Result】The strain D6 with the fastest mycelium growth rate（0.477 cm/d） was selected, the colony edges were neat, the mycelia were dense, and it was CK. The polymorphism rate of 6 primers with clear bands and good polymorphism was 88.5%. When the genetic similarity coefficient was 0.73, the 10 strains could be clustered into 3 categories. The protoplasts of the CK strain were mutated by atmospheric and room temperature plasma, and 6 dominant strains with faster mycelium growth rate and obvious antagonism with CK were selected. There was no obviouis difference in mycelium growth rate of the same mutagenic strains at different subcultures, CK had the lowest mycelium growth rate and strain D32 had the highest mycelium growth rate, which was significantly higher than that of other strains under the same subculture condition（P＜0.05）. Genetic difference analysis showed that there were genetic differences between the 6 dominant strains and CK, and the nucleic acid sequences were changed compared with CK. 【Conclusion】The genetic basis of 10 S. rugosoannulata strains is concentrated, and 6 strains with obvious and stable mutagenesis effect are selected, which are D32, D36, D42, D73, D118 and D133 respectively, they can be used for further research on new S. rugosoannulata variety breeding.