Objective: To establish a DNA barcoding based method for guarantee the species authenticity of Anemarrhenae Rhizoma seeds. Method: Thirty original plant materials and nine raw materials have been collected for obtaining the reference DNA barcode sequences.The clone sequencing technique was employed to confirm the reliability of reference sequences.Fifty-one samples of Anemarrhenae Rhizoma seeds pending to test have been collected

and DNA barcodes of these samples were obtained for taxonomic assignment by BLAST method

genetic distance method and neighbor-joining(NJ) phylogenetic tree method. Result: For the original plants and raw materials

their internal transcribed spacer 2(ITS2) sequences could not be obtained stably as a result of intra-genomic heterogeneity.Meanwhile

their psbA-trnH sequences could be successfully obtained.Their psbA-trnH sequences could be divide into six haplotypes

including three variation sites and two insertions/deletions.The maximum intra-specific distance was 0.003 5

whereas the minimum inter-specific distance was 0.1.Their psbA-trnH sequences were composed of one clade at the NJ dendrogram.There were 153 psbA-trnH sequences have been obtained and all can be assigned to be Anemarrhena asphodeloides with BLAST method

genetic distance method and NJ phylogenetic tree method. Conclusion: The DNA barcoding technology is a reliable method for species identification of Anemarrhenae Rhizoma seeds.All the samples of Anemarrhenae Rhizoma seeds pending to test are in accordance with the origin of that ruled on the 2015 edition of Chinese Pharmacopoeia and no adulterant has been detected.