To identify C.japonicum, C.setosum and their adulterants through ITS2 Sequence method to ensure clinical medication safety. Methods:DNA was extracted from C. japonicum and C.setosum, which were the original plant samples of Cirsii Japonici Herba and Cirsii Herba together with their adulterant and ITS2 sequences were obtained by PCR, sequencing, and sequence assembly. Totally, 54 sequences of 14 species were downloaded from the GenBank, using MEGA 6.0 to align all sequences, calculating the intraspecific and interspecific Kimura-2-Parameter (K2P) genetic distance and constructing NJ tree. Results:It suggested that the intraspecific variation locis between C.japonicum and C.setosum were much less than that between adulterants. The maximum (average) intraspecific distance of C. japonicum and C.setosum was lower than the minimum (average) interspecific distance of adulterants. Based on ITS2 sequences, this identification method of NJ Tree were effective and clearly distinguished C. japonicum and C.setosum from their adulterant. Also, all species showed obvious monophyly. Conclusion:The sequence of ITS2 identified the herbal materials of C.japonicum, C.setosum and their Adulterants.