Objective: To establish a method for indentification of the podophyllum emodi species, Radix Clematis and Radix Gentiana species. Methods: HPLC/UV fingerprint analysis method of toxic ingredient podophyllotoxin and its derivatives were developed, and the method had been evaluated. Results: The methodological evaluation showed that this method had a good repeatability and reproducibility, and different samples had different HPLC fingerprints. Conclusion: This method can be used to differentiate podophyllum emodi from two commonly used medicinal herbs of a different genus but having similar appearance, Radix Clematis and Radix Gentiana.