Abstract: Abstract: SSR primers of Lentinula edodes were developed using ISSR-suppression PCR method and Primer 3 program, and L16（45）orthogonal design was used to optimize main factors affecting SSR-PCR amplification system. The optimal SSR-PCR system was established as follows, within 20μl reaction system, there were 1.5 mmol/L Mg2+, 75 ng template DNA, 0.25 mmol/L dNTPs, 0.50 μmol/L primer and 1.5U Taq DNA polymerase. The optimal annealing temperature was determined as 62℃ by gradient PCR. Based on the optimal system, suitable results were obtained when five primer pairs were used to amplify genomic DNA of eight strains .Cluster analysis well reflected geographical original relation of these strains. The eight strains were classified into two groups using 0.5 similarity as a cut-off point. GroupⅠmainly consisted of strains from North China, while group Ⅱ comprised those from South China.