Abstract: Herba Anoectochili is a commonly used medicinal material. However, its adulteration is a serious concern. Due to the similar morphological characteristics of Herba Anoectochili and its adulterants, traditional identification techniques often fail to distinguish between them accurately, which is not conducive to the circulation management and safety of the medicinal materials. To improve the distinction between Herba Anoectochili and its adulterants accurately, this study identified 41 Herba Anoectochili and its adulterant samples based on the ITS2 sequence. Sequence characteristics, Basic Local Alignment Search Tool (BLAST) application, genetic distance, construction of phylogenetic tree, secondary structure prediction, and other methods showed the ITS2 sequence to accurately identify Herba Anoectochili from its adulterants. Furthermore, in this study, we designed a specific primer, based on the ITS2 sequence, and established a real-time PCR detection system for the rapid, sensitive, and specific identification of the original plant of Herba Anoectochili. Compared to DNA barcoding technology, this method has shorter detection time, stronger specificity, and higher sensitivity, which lays the foundation for the rapid identification of Herba Anoectochili.