We aimed to establish a rapid and accurate allele-specific diagnostic Polymerase Chain Reaction (PCR) method for medicinal pipefish.To achieve this,pipefish genomic DNA was extracted,sequenced bi-directionally,and the data were analyzed.On this basis,specific identification primers were designed and a facile multiplex PCR system was established and optimized.Phylogenetic tree analysis showed that the six species of pipefish were strictly clustered in separate single branches.