Abstract: OBJECTIVE To provide a new method for the identification of Rubi Fructus in medicial market based on ITS2 barcode, and guaranting the accuracy and safety of Rubi Fructus. METHODS Combined with the sequences downloaded from GenBank, a total of 48 ITS2 sequences of Rubus chingii Hu and its confusable species of genus Rubus were obtained. Sequence characteristics such as length, GC contents, and mutation loci were analyzed through Gene Tool software, while genetic distance calculation and build-up of neighbor-joining(NJ) phylogenetic tree were carried out by Clustal X and MEGA 7.0. Simultaneously, 24 samples of Rubi Fructus collected from markets randomly were detected by Chinese herbal medicine DNA barcoding identification system and build-up of NJ phylogenetic tree. RESULTS The results revealed that R. chingii Hu and its adulterants could be differentiated obviously. The 22 samples of Rubi Fructus collected from markets were genuine, 1 sample was counterfeit and 1 sample was mixture. CONCLUSION The DNA barcoding technology based on ITS2 sequences can identify R. chingii Hu from its adulterants successfully.