The Chinese caterpillar fungus Ophiocordyceps sinensis, endemic to alpine regions on the Tibetan Plateau, is one of the most valuable medicinal fungi in the world. Genetic differentiation within this fungus was observed; however, due to lack of highly efficient molecular markers, the overall genetic structure of this fungus has not been clarified. In this study, a shotgun genomic library of O. sinensis was constructed, and > 181,848 nt were analyzed from > 250 random clones. Primers from 33 sequenced fragments were then designed to amplify O. sinensis samples collected from widely separated regions on the Tibetan Plateau. Ten of the 33 fragments had no amplification or poor sequencing quality from all or certain samples. Sequence variations of the remaining 23 fragments among different samples were investigated in detail. Three fragments (OSRC14, OSRC19, and OSRC32) were the most variable with 7-43 single-nucleotide polymorphism (SNP) sites, representing the SNP frequency of 1.2-6.7 % per nucleotide site. These three fragments have the potential to be useful molecular markers for studying the population genetics of O. sinensis. These results also showed that constructing and screening a shotgun genomic library was an efficient approach to identify novel molecular markers from non-model organisms.