Abstract: Seven cultivated strains of Pleurotus eryngii were used as experimental materials to identify and classify different strains by antagonistic test, esterase isoenzyme and ISSR molecular marker technology, using protoplast monokaryons hybridization technology, different species and different mating types of strains were selected for mon-mon hybridization.The hybrids with clamp connection were selected, and the mycelium growth rate and agronomic traits of hybrid strains and parental strains were compared, in order to provide excellent germplasm resources for the actual production and cultivation of Pleurotus eryngii.The results showed that the seven strains were divided into three groups, the first group was 12,16,1208,1219 and 1287 strains.The second group was 1283 strain.The third group was 1284 strain.Twenty-four hybrids were obtained by protoplast monokaryons hybridization.Compared with the parents, five hybrids with significantly better mycelium growth rate, agronomic traits were selected, namely ZJ5,ZJ8,ZJ11,ZJ17 and ZJ22,respectively.