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Tongren Dahuoluo Wan has been a popular traditional Chinese medicine in international pharmaceutical markets for hundreds of years. Leopard bone powder is the key element in its formulation. However, the leopard has been listed for wildlife conservation, which limits the use of the leopard bone supplies. Therefore, an alternative formulation which substitutes leopard bone with zokor bone in the formula of Tongren Dahuoluo Wan is now manufactured. To develop a simple and reliable molecular method for authenticating the two patent medicines, mitochondrial nucleotide polymorphic sites of 12S rRNA, COI and Cytb genes were screened in leopard and zokor bones, and nine pairs of species-specific primers were verified for discriminating the two species. For the patent medicine authentication, we set up a molecular diagnostic assay to resolve the difficulties of low concentration of target DNAs and presence of PCR-inhibitory substances in this complex medicine, and successfully confirmed leopard or zokor content using the nine pairs of species-specific primers. We recommend a common technical strategy for authentication of species origins in traditional Chinese medicine, and discuss the experimental solutions for technical problems of molecular diagnostic assays.