Abstract: OBJECTIVE To establish a polymerase chain reaction(PCR) method for rapid interspecific identification of Hirudo.METHODS Three pairs of specie-specific primers were designed and synthesized according to the differences of CO1 sequences of three kinds of Hirudo(Whitmania pigra Whitman, Hirudo nipponica Whitman, Whitmania acranulata Whitman) in the Chinese Pharmacopoeia, and the PCR reaction conditions were optimized. The stability experiment was carried out for different batches of Hirudo, and the sensitivity experiment was carried out for the same batch of Hirudo. The optimized PCR conditions were used to analyze the mixed Hirudo samples.RESULTS The 3 pairs of primers could amplify the bright bands of 1 000-2 000, 1 000-2 000 and 3 000 bp at annealing temperature of 60, 61 and 59℃ respectively, with detection limit of 0.1, 1 and 10 ng respectively, and could accurately identify the Hirudo samples mixed in different proportions.CONCLUSION The 3 pairs of primers can be used for the rapid identification of Hirudo, common counterfeit and mixed Hirudo with high interspecific specificity and good intra-specific stability and sensitivity, which can be used as a supplement to the traditional identification method of Hirudo and provide more reference for its quality control.