Ophiocordyceps sinensis (Berk.) G.H.Sung, J.M.Sung, Hywel-Jones & Spatafora, an endangered ascomycete fungus endemic to the Tibetan Plateau, is one of the most important traditional Chinese medicines with a huge economic value and has been used to treat a wide range of conditions.In this study, an ISSR-TA1L-PCR method, which directly combines inter-simple sequence repeat (ISSR) and thermal asymmetric interlaced PCR (TAIL-PCR) technologies without traditional DNA library construction and screening with appropriate probes, was established to develop microsatellite markers for O.sinensis.It is a simple strategy for microsatellite development and may be applied to other taxa.A total of 30 perfect and imperfect microsatellites were identified in 48 individuals of O.sinensis from five provinces within China representing different populations.Seventeen loci were polymorphic with two to four alleles per locus, while 13 were monomorphic.The microsatellite markers developed here may be used in studies of population genetics and conservation biology of O.sinensis.Furthermore, specific primer pairs were designed based on a comprehensive ITS sequence dataset of O.sinensis and its related fungi, and tested for specificity and sensitivity through PCR experiments using 27individuals of O.sinensis from different geographical origins and 40 other related fungal species in terms of phylogeny or ecology.A primer pair highly specific to O.sinensis was obtained, yielding a 275 bp PCR product from all the individuals of O.sinensis but no product from the other fungi tested.The detection limit of the primers was demonstrated to be 10 ng of pure O.sinensis DNA for conventional PCR and 10 pg for nested PCR in a 25 tμ1reaction system.Soil samples collected from the habitat of O.sinensis were also tested using this PCR assay.The results showed that the primer pair and PCR-based assays developed in this study can be applied to the rapid detection of O.sinensis in its natural habitat.