DNA barcoding is a novel technique which enables species identification and taxonomic classification using a short and standard DNA sequence. In this study, traditional herbal medicine Nervilia fordii and its congeneric species included all three distributed in mainland China and three acquired from Genbank were sampled. Four popular candidate DNA barcodes, ITS2, rbcL, matK, and LSU D1-D3, were amplified and sequenced with corresponding pairs of universal primers. The PCR amplification and sequencing efficiency, intra-and interspecific variation and barcoding gap were used to evaluate each locus, and the success rate of correct identification was assessed by the BLAST 1 method. The sequencing efficiency of rbcL and LSU D1-D3 was 100%, while that of matK and ITS2 was 95.8% and 91.4%, respectively. Although relatively lower contribution in sequencing efficiency and interspecific divergence, matK exhibited significant barcoding gap and discriminated all the given species in our study. ITS2 had the largest variation in both intra-and interspecific level, which enabled classification at the genus level. Our findings showed that DNA barcoding is an efficient biomarker for identification of N. fordii and its congeners and that matK gene is a potential DNA barcode for Nervilia. Abbreviation: CBOL-the Consortium for the Barcode of Life; ITS2-the second internal transcribed space; LSU-ribosomal RNA large subunit gene; matK-maturase K; NCBI-National Center for Biotechnology Information; rbcL-ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit.