DNA barcoding is a rapidly developing frontier technology in the world and will be useful in promoting the quality control and standardization of traditional Chinese medicine.  Until now, many studies concerning DNA barcoding have focused on leaf samples but rarely on Chinese herbal medicine.  There are three issues involved in DNA barcoding for traditional Chinese medicinal materials: ① the extraction methods for total DNA of the rhizomes of the medicinal materials; ② intra-specific variation among samples from different places of origin; ③ accuracy and stability of this method.  In this study, Gentianae Macrophyllae Radix was used to verify the stability and accuracy of DNA barcoding technology.  Five regions (ITS2, psbA-trnH, matK, rbcL, and ITS) were tested for their ability to identify 86 samples of Gentianae Macrophyllae Radix and their adulterants.  After improving the DNA extraction method, genomic DNA from all samples was successfully obtained.  To evaluate each barcode’s utility for species authentication, PCR amplification efficiency, genetic divergence, and species authentication were assessed.  Among all tested regions only ITS2 locus showed 100% of PCR amplification and identification efficiencies.  Based on the established method, we successfully identified two samples of Gentianae Macrophyllae Radix bought in pharmacy to the original species.