Abstract: To accurately discriminate Stellariae Radix from its adulterants, four leading candidate DNA barcoding markers were evaluated. Sixty samples including Stellariae Radix and its adulterants have been newly collected and their total genomic DNA was extracted. Four DNA barcoding markers ITS, rbcL, psbA-trnH and matK were amplified and sequenced. Their sequence characteristic analyses, Kimura-2-parameter (K2P) distance calculation and Neighbor-joining (NJ) phylogenetic tree constructions were accomplished using the MEGA 7.0 software. DNA Barcoding gaps of the four DNA barcoding markers were estimated by the distributions of interand intra-sequence specific variations. Species identification efficiency was calculated using the BLAST method. The results showed that ITS had the highest (95.2%) while matK demonstrated the lowest (75%) PCR and sequencing efficiency. The length range of the four markers were in the ranger of 211-797 bp, and the G+C content of ITS was highest (54.35%). The identification efficiency of matK and ITS was 92% and 90% respectively. Barcoding gap could be found in ITS sequences. The NJ phylogenetic tree constructed using ITS sequences showed that samples of Stellariae Radix were separately formed into one clade, and samples of adulterants like Stellaria bistyla were clearly belong to different branches from Stellariae Radix, whereas NJ trees constructed using psbA-trnH, rbcL and matK could not differentiate Stellariae Radix from its adulterants. Therefore, ITS regions as DNA barcodes can stably and accurately distinguished Stellariae Radix from its adulterants, and provide a new technique for modern identification of Stellariae Radix.