[Objective] To establish molecular identification method for Ilex asprella var. asprella and relative medicinal plants in the genus Ilex base on ITS2 sequence. [Method] Total DNA was extracted and a pair of universal primers were used for PCR amplification and sequencing of ITS2 region. The sequences of different samples were annotated by HMMer method to acquire ITS2 sequences. A phylogenetic tree was constructed using the software MEGA, based upon the multiple alignment of the amplified ITS2 sequences. [Result] 254 bp ITS2 fragment of each species were obtained, and 85 different base sites between species were found. [Conclusion] ITS2 region can be used to identify Ilex asprella var. asprella from relative medicinal plants in the genus Ilex. [Objective] To establish molecular identification method for Ilex asprella var. Asprella and relative medicinal plants in the genus Ilex base on ITS2 sequence. [Method] Total DNA was extracted and a pair of universal primers were used for PCR amplification and sequencing of ITS2 region . The sequences of different samples were annotated by HMMer method to acquire ITS2 sequences. A phylogenetic tree was constructed using the software MEGA, based upon the multiple alignment of the amplified ITS2 sequences. [Result] 254 nt ITS2 fragment of each species were obtained, and 85 different base sites between species were found. [Conclusion] ITS2 region can be used to identify Ilex asprella var. asprella from relative medicinal plants in the genus Ilex.