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The CO I gene sequences of Qianghuoyu, Pachytriton labiatus and Gehyra mutilata were achieved by PCR amplification and bi-directional sequencing. Furthermore, a pair of specific primers SJYW1 and SJYW2 in the non-conservative district were designed through sequence alignment. The PCR reaction condition was established by changing the annealing temperature and cycle numbers. The results showed that 350 bp DNA fragment was amplified from Qianghuoyu in PCR with annealed temperature at 54 °C and the cycle number was 25 cycles, whereas not any DNA fragment was amplified from P. labiatus and G. mutilata under the same reaction condition. This method is well-performed in the identification of Qianghuoyu for its excellent specificity and repeatability.