Pleurotus eryngii (DC. Ex. Fr.) Quél is a rare precious edible fungus which belongs to the family Pleurotaceae. This mushroom has highly nutritional, pharmaceutical, economic and ecological values. In the present study, combined randomly amplified polymorphic DNA (RAPD)/inter-simple sequence repeat (ISSR) was used to assess the genetic diversity of P. eryngii strains cultivated in China. For the RAPD and ISSR analyses, 404 and 392 polymorphic bands were obtained from 32 P. eryngii strains using 28 and 24 selected primers, respectively. A combined RAPD/ISSR dendrogram grouped the 32 strains into five clades with coefficient of 0.770. The comparison of RAPD and ISSR was also elucidated in the present study. The results of our study obtained by combined RAPD/ISSR analysis contributed to a better understanding of the genetic relationships among the P. eryngii strains and provide orientation for the strain improvement of P. eryngii species.