AIM: An efficient, precise, and sensitive method for identifying Atractylodes plants has been established and will contribute significantly to quality control and scientific analysis in Chinese traditional medicine. METHODS: Twenty primers were applied for setting up the RAPD (randomly amplified polymorphic DNA) markers of Atractylodes plants, Atractylodes lancea DC (A lancea DC), Atractylodes japonica Koidz (A japonica K), and Atractylodes ovata DC (A ovata DC). The primer OPF03, OPF05, and OPF14 could discriminate them successfully. The results were also able to apply on the Chinese formulations with Atractylodes purchased from local markets. RESULTS: RAPD was used to investigate phylogenetic relationships among and within closely related species. RAPD analysis reflects heritable changes in the nucleotides sequence in both the coding and noncoding regions, because it is conducted directly from the DNA level. This work first conducted RAPD analysis of Atractylodes plants to establish their RAPD makers. CONCLUSION: The RAPD markers could be applied extensively in the Chinese herbal formulations.