To establish a method for the molecular authentication of Fallopia multiflora.The trnL-trnF regions of Fallopia multiflora and its closely related species and/or adulterants were sequenced and analyzed.It was found that the trnL-trnF sequence divergences between Fallopia multiflora and its closely related species and/or adulterants were 2.1%-22%. While the intra-species trnL-trnF divergences of Fallopia multiflora were 0%-1.5%. Based on the trnL-trnF regional variations, an endonuclease Xba I (T CTAGA) restriction site specific to Fallopia multiflora was detected. The Fallopia multiflora trnL-F polymerase chain reaction product could be cleaved by Xba I into two pieces, 804-819 bp and 256 bp each, whereas the restriction endonuclease could not digest the trnL-trnF polymerase chain reaction product of its closely related species or adulterants. The restriction patterns analyzed for restriction enzyme Xba I were found to be identical in all Fallopia multiflora individuals from different geographical regions in China.The assay based on polymerase chain reaction amplification of the trnL-trnF fragment of chloroplast DNA and subsequent restriction fragment length polymorphism can be used as a general test to identify Fallopia multiflora.