A gradient reversed-phase high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination of seven nucleosides, adenosine, cordycepin (3'-deoxyadenosine), cytidine, guanosine, thymidine, uridine, and inosine, and five nucleobases, adenine, cytosine, thymine, uracil, and hypoxanthine in Cordyceps sinensis, Cordyceps militaris, Ganoderma lucidum, Agrocybe aegerita, Termitornyces albuminosus, and Lentinus edodes. The results showed that total nucleoside and nucleobase contents ranged from 0.14 to 26.57 mg/g dry matter in these fungi. The higher total nucleoside and nucleobase levels (>10 mg/g dry matter) were found in the gills and the pilei of A. aegerita and T. albuminosus, and the gills of L. edodes. The lower levels (<1 mg/g dry matter) were detected in the stipe and the pileus of G. lucidum. The results indicated that A. aegerita, T. albuminosus, and L. edodes had much higher contents of nucleosides and nucleobases than C. sinensis, C. militaris, and G. lucidum. It is notable that the hymenophore tissues, which contained the spore-producing cells, such as the gills for A. aegerita, T. albuminosus, and L. edodes, the tubes for G. lucidum, and the perithecia for C. sinensis, were found to have considerably higher amounts of total nucleosides and nucleobases as compared to other parts of these fungi.