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In this study, the species identifications of seven Ephedra plants, including three medicinal plants from the Pharmacopoeia of the People’s Republic of China, were conducted using phylogenetic analyses, and the method’s validity was verified. The phylogenetic trees constructed from the maturase-coding gene (matK) and internal transcribed spacer 1 (ITS1) sequences showed that the former could be used for identifying five Ephedra plants, Ephedra intermedia, E. equisetina, E. antisyphilitica, E. major, and E. aphylla, but it had less power to discriminate E. sinica and E. przewalskii, while the latter could distinguish five Ephedra plants, E. przewalskii, E. equisetina, E. antisyphilitica, E. major, and E. aphylla, but it had less power to discriminate E. sinica and E. intermedia. However, when the two genes were combined, the seven species could be completely distinguished from each other, especially the medicinal plants from the others, which is significant in developing their pharmaceutical uses and in performing quality control assessments of herbal medicines. The method presented here could be applied to the analysis of processed Ephedra plants and to the identification of the botanical origins of crude drugs. Additionally, we discovered that E. equisetina and E. major were probably closely related to each other, and that E. sinica, E. intermedia, and E. przewalskii also had a close genetic relationship.