Abstract: It was to establish a novel identification method for Nervilia fordii and its adulterants through ITS2 sequence analysis. TotalDNA was extracted from fresh leaves, and ITS2 region was amplified and sequenced with a pair of universal primers. The sequences wereassembled and aligned by DNAMAN and CLUSTALX, and NJ tree was constracted using MEGA 4.0. Secondary structures of ITS2 sequenceswere predicated in the database built by Schultz et al. Significant differences were observed in the ITS2 sequences between Nervilia fordii andits adulterants. The interspecific genetic distance was far more than the intraspecific disatance. Both NJ tree and secondary structure based onITS2 sequences can distinguish Nervilia fordii and its adulterants intuitively. ITS2 is an effective biomarker to identify Nervilia fordii and itsadulterants.