To develop a method to identify Clematidis radix et Rhizoma using sequence similarity and sequence-specific genetic polymorphisms based on the ITS sequences. DNA was extracted from leaves of Clematis mandshurica Rupr and C. hexapetala using a DNA extraction kit. ITS sequences were amplified by PCR, and analyzed in Contig Express, DNAman, and MEGA 5.0. The core haplotype was determined, and similarities between the core and other haplotypes were calculated. In total, 138 ITS sequences of C. mandshurica were obtained with a length of 611 bp. The similarity threshold between C. mandshurica and counterfeit species was 99%. Using specific mutation sites, we could identify C. chinensis, C. hexapetala, and C. mandshurica rapidly and accurately. A new DNA-based method has been established to rapidly and accurately identify Clematidis radix et Rhizoma.