Background: Peucedanum praeruptorum L., a traditional Chinese medicine known as Qian-hu, is commonly used for dispelling wind-heat and expectorant and loss of energy. However, due to similar morphological characters and high market demand, there are many substitutes and adulterants of P. praeruptorum. DNA barcoding is an approach to identify species based on sequences from a short, standardized DNA region. Objective: To authenticate P. praeruptorum from its substitutes and adulterants. Materials and Methods: The differential identification of P. praeruptorum and 13 regional substitutes and 23 adulterants was investigated by means of DNA sequence analysis of internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (nrDNA), a bootstrap neighbor-joining (NJ) tree according to Kimura's 2-parameter method was also calculated. Results: The data showed that P. praeruptorum, its substitutes and adulterants could be easily distinguished at the DNA level, while almost all species were well resolved, and successfully identified on the NJ tree. Conclusion: The ITS sequence can be used for the identification of P. praeruptorum and to distinguish it from common substitutes and adulterants.