Abstract: In this study,three specific primers pairs were designed bases on comparison of the difference of the ITS gene sequences of Rhizoma Paridis’ legal origins (Paris polyphylla var. chinensis and P.polyphylla var.yunnanensis) from their closely related species.A fast and accurate method was established to identify Rhizoma Paridis’ legal origins and their closely related species according to the principles of multiplex polymerase chain reaction(PCR).Eventually,the size of 210 bp specific bands were amplified from the P.polyphylla var. chinensis;the sizes of 248 bp and 149 bp specific bands were amplified from P.polyphylla var. yunnanensis samples of two kinds of genotypes (YN-I and YN-II) respectively.The positive identification of legal origins were obtained while their closely related species weren’t amplified any bands to interfere with the result.For the first time,this method realized authentification of Rhizoma Paridis’ legal origins in one multiple PCR identification system simultaneously.This method is of great significance for improving the technical level of quality control of Rhizoma Paridis.

Key words: Rhizoma Paridis, legal origins, closely related species, multiplex polymerase chain reaction, identification, Paris polyphylla var. chinensis, Paris polyphylla var.yunnanensis