Morus Folium (Sang-yeop in Korean) is one of the most important Oriental medicinal plants. In Korea, both M. alba and M. cathayana are regarded as the botanical sources for Morus Folium. In order to discriminate M. alba and M. cathayana from their adulterant, M. tricuspidata, mitochondrial NADH dehydrogenase subunit 7 (nad7) intron 2 region was targeted for molecular analysis with universal primers. DNA polymorphisms, including SNP sites, insertions, and deletions, were detected among these three species sequencing data. Based on these DNA polymorphisms, specific primers were designed for the three species respectively. Multiplex PCR was conducted for molecular authentication of M. alba, M. cathayana, and M. tricuspidata with specific primers. The present results indicate that it is possible to identify Morus Folium from its adulterant using mitochondrial nad7 intron 2 region. The established multiplex-PCR system was proved to be effective for identification of Morus Folium. The results indicate that mitochondrial introns can be used for inter-specific polymorphic study, and the described method can be applied for molecular identification of medicinal materials.