Abstract: 【Objective】 This study aimed to analyze the genetic diversity and conduct a comprehensive evaluation of Stropharia rugosoannulata Farl. ex Murrill germplasms based on ISSR molecular markers and phenotypic traits， and to screen phenotypic traits and excellent germplasms for identification and evaluation， providing a scientific basis for the exploitation and utilization of Stropharia rugosoannulata Farl. ex Murrill germplasm resources.【Method】 The genetic diversity of 47 Stropharia rugosoannulata Farl. ex Murrill germplasms was analyzed by ISSR molecular markers， and 15 phenotypic traits were determined to calculate their Shannon-Weaver diversity indexes and coefficients of variation for each trait. Correlation analysis， cluster analysis， principal component analysis， and stepwise regression were employed to perform a comprehensive evaluation of the germplasms.【Result】 A total of 106 polymorphic bands were amplified by 18 ISSR primers， with an average polymorphism percentage of 68.03%. The genetic similarity coefficients of 47 Stropharia rugosoannulata Farl. ex Murrill germplasms were 0.80-0.99. The 47 Stropharia rugosoannulata Farl. ex Murrill germplasms were divided into five major clusters at the genetic similarity coefficient of 0.896. The pileus shape， pileus scale number， pileus color， and initial harvest period were identified as the primary characteristic traits for ISSR-based classification. The Shannon-Weaver diversity indexes of the 15 phenotypic traits were 0.18-1.90， with an average value of 1.03. Among them， the Shannon-Weaver diversity indexes of six phenotypic traits （stipe diameter， pileus diameter， pileus height， pileus color， pileus thickness， and stipe length） were greater than 1.00， indicating a high degree of variation. The coefficients of variation for five measured traits of fruiting bodies （pileus height， pileus diameter， pileus thickness， stipe length， and stipe diameter） were 24.20%-32.29%， all higher than 20.00%， indicating a high degree of variation. Extremely significant correlations were found among 17 pairs of phenotypic traits （P<0.01， the same below）， of which 14 pairs showed extremely significant positive correlations. All five measured traits of fruiting bodies exhibited extremely significant positive correlations with each other （P<0.01）. The cluster analysis （type-y） of Stropharia rugosoannulata Farl. ex Murrill germplasms based on phenotypic traits showed that 47 germplasms were divided into five major clusters. The pileus shape， pileus scale number， pileus color， stipe shape， and initial harvest period were identified as the primary cha-racteristic traits for cluster classification. Principal component analysis results showed that the cumulative contribution rate of the first six principal components reached 77.66%， which could summarize the main information of phenotypic traits of Stropharia rugosoannulata Farl. ex Murrill. The comprehensive scores of principal components （F values） of 47 germplasms ranged from -1.99 to 1.48， based on which they were divided into six clusters. The five characteristic traits for cluster classification were pileus shape， pileus scale number， pileus color， stipe shape， and stipe color， the first four of which were the same as those of the type-y cluster classification. The six principal components were analyzed through the membership function analysis and a mathematical model for comprehensive evaluation was obtained： F=0.31F₁+0.13F₂+0.11F₃+0.08F₄+0.08F₅+0.07F₆.【Conclusion】 ISSR molecular markers can be used to analyze the genetic variation among Stropharia rugosoannulata Farl. ex Murrill germplasm resources. Based on the F values， six germplasms with excellent phenotypic traits are selected， which can be developed and utilized as breeding materials. Six phenotypic traits （pileus shape， pileus scale number， pileus color， stipe shape， stipe length， and initial harvest period） are suitable indexes for comprehensive evaluation of Stropharia rugosoannulata Farl. ex Murrill germplasms.