To identify Ejiao and its adulterants at the DNA level by using DNA molecula marker. Ejiao (Asini Corii Colla) is a commonly used medicinal material. However, its adulteration is a serious concern. Due to the morphological characteristics of Asini Corii Colla and its adulterants, traditional identification techniques often complex and professional, which is not conducive to the circulation management and safety of the medicinal materials. To improve the distinction between Asini Corii Colla and its adulterants accurately, this study identified and its adulterant samples based on the CytB sequence. Sequence characteristics, Basic Local Alignment Search Tool (BLAST) application, genetic distance, construction of phylogenetic tree showed the CytB sequence to accurately identify Asini Corii Colla from its adulterants. Furthermore, in this study, we designed a specific primer, based on the CytB sequence, and established a PCR detection system for rapid, sensitive, and specific identification of Asini Corii Colla. 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 Asini Corii Colla.