Dyers woad leaf (Daqingye) is a traditional Chinese medicine commonly used as anti-pyretic,anti-bacterial and anti-viral agent against infectious diseases. The Chinese Pharmacopoeia (2005)records that Dyers woad leaf should be derived from the leaves of Isatis indigotica Fort., but theleaves of Polygonum tinctorium Ait., Baphicacanthus cusia (Nees) Bremek. and Clerodendron cyrtophyllumTurcz. have also been used as substitutes of Dyers woad leaf in different regions of China. The leafmorphologies of these four species show a close resemblance, and based on their morphologicalappearance, it is difficult to identify them. Here, molecular genetic methods were developed as atarget to identify different members of Dyers woad leaf. The 5S-rRNA spacer domain wasamplified by polymerase chain reaction (PCR) from genomic DNAs isolated from I. indigotica, P.tinctorium, B. cusia and C. cyrtophyllum, and the nucleotide sequences showed a great diversity. Inaddition, random amplification of polymorphic DNA (RAPD) analysis was also used todistinguish the members of Dyers woad leaf. These molecular methods could be used as a tool inauthentic identification of Dyers woad leaf.