Three difficultly-divisional species, i. e., Ainsliaea fragrans, A. macroclinidioides and A. rubrifolia were studied using the ISSR molecular identification system. Twenty primers were screened out from 100 ISSR UBC. They could generate reproducible polymorphic fragments. The particular bands were expanded to make the ISSR fingerprint by primer UBC807, UBC826, UBC834, UBC843, UBC853, and UBC895. It could be used to discriminate three species. Furthermore, according to the alike coefficient UPGMA showed that the re]ative relation of A. fragrans and A. rubrifolia was closer than that of A. fragrans and A. macroclinidioide. The research could be responded for the conservancy and development of the chinese herbal medicine resonrce, and providing a technical support of building up the stable qnality evaluation system of chinese herbal medicine.