Abstract: OBJECTIVE To establish an HPLC method for determining the contents of five nucleosides (namely uracil, hypoxanthine, uridine, guanosine and inosine), and compare the content differences of the five nucleosides in pilos antler from Cervus nippon and Cervus elaphus. METHODS The HPLC method was applied to determine 15 batches of pilos antler from Cervus nippon and Cervus elaphus, and a cluster analysis was carried out for further results. RESULTS The contents of five nucleosides in pilos antler from Cervus nippon and Cervus elaphus were tested. There were significant differences in contents of uracil, hypoxanthine, uridine, guanosine and inosine for pilos antler from Cervus nippon and Cervus elaphus; there were significant differences in contents of the five nucleosides for pilos antler from different farmers of a same region. Fifteen batches of pilos antler from Cervus nippon and Cervus elaphus were divided into two categories by clusteranalysis. CONCLUSION The contents of five nucleosidess indicates significant difference from Cervus nippon and Cervus elaphus. The quality of the pilos antler is affected by variety of deer, natural environment and technical factors of farming. The method is suitable for the determination of nucleotides content in the pilos antler from Cervus nippon and Cervus elaphus, and provides guarantee for their quality control.