Abstract: In order to clearly preserve the relationship between the germplasm resources of Lentinula edodes, the new varieties with independent intellectual property rights and suitable for cultivation in western Inner Mongolia were selected to provide a basis. The polymorphism of 26 Lentinula edodes isolates was detected by using ISSR molecular marker and 10 primers. Cluster analysis and genetic diversity comparison were performed by NTSYS software. The results showed that 70 stable bands and 67 polymorphic bands could be amplified with 10 pairs of primers, the polymorphism ratio was 95.71%, and the range of genetic distance ranged from 0 to 0.749 9. When the genetic similarity coefficient was 0.678, the strains were divided into 4 groups. ISSR molecular marker technique could effectively reveal the genetic diversity of germplasm resources of 26 Lentinula edodes strains and the relationship between them.