In this study,we used Pleurotus.nebrodensis,Pleurotus.eryngii and Pleurotus.ferulae as tested materials to extract the mitochondrial DNA of the tested strains with the method of differential centrifugation and improved CTAB,and applied the extracted DNA as template,MS1 and MS2 as specific primers to amplify the DNA fragment of tested strains mitochondrial SSU rDNA 5′ PCR products and then they were purified and sequenced.After being proofread,the sequence length GC content and genetic distance of the measured sequences were analyzed with the BioEdit 6.0.5 and MEGA 3.1 software to determine the genetic relationship of the tested strains.The result showed that Pleurotus.nebrodensis,Pleurotus.eryngii and Pleurotus.ferulae have the close genetic relationship,and the genetic relationship between the Pleurotus.ferulae and Pleurotus.nebrodensis was much closer,but the Pleurotus.nebrodensis,Pleurotus.eryngii and Pleurotus.ferulae belonged to three different species.And the results also showed that the mt SSU rDNA 5′ sequence can be used to distinguish different species.