Abstract: OBJECTIVE The aim of this study was to identify Alisma orientale (Sam.) Juzep. and its adulterants using the ITS2 barcode. METHODS The total DNAs were extracted from twenty-eight samples of A. orientale as well as adulterants. The ITS2 sequences of these samples were amplified and sequenced. The acquired sequences were submitted to the GenBank and the other ITS2 sequences of 17 samples from ten species were downloaded from the GenBank. Total 45 ITS2 sequences were aligned and the genetic distances among them were analyzed with MEGA 5.1. The sequences analyses were performed using the BLAST1 and the nearest distance methods， and were presented intuitively by constructing Neighbor-joining (NJ) tree. RESULTS The lengths of all ITS2 sequences of A. orientale and A. plantago-aquatica were 311 bp. Only one mutation existed among them. There was significant divergence between the interspecific and intraspecific genetic distances of the ITS2 sequences. The NJ tree showed that A. orientale is obviously different from its adulterants， which showed high monophyly. CONCLUSION As a DNA barcode， ITS2 sequences can stably and accurately distinguish A. orientale from its adulterants and also provide a new clue to identify the medical germplasm resources and ensure the clinical safety in utilization of Chinese materia medica.