The two main secondary metabolites in Glycyrrhiza Species are Glycyrrhizic acid and liquiritin. They are considered as active ingredients . The content of these compounds showed variation in different species. Standard chemical fingerprints were generated from cultivated Glycyrrhiza uralensis, G. inflate and G. glabra, which could be identification markers. Five efficient inter-simple sequence repeat (ISSR) primers were screened and optimized for detecting the genetic diversity in three cultivated Glycyrrhiza uralensis, G. inflate and G. glabra. By using two characteristic peaks compare with three cultivars, Glycyrrhiza uralensis and G. glabra were bigger similarity than G. inflate. The results is in accordance with the results by ISSR markers. The higher genetic diversity in G. inflate was useful to more broad breeding. Our result suggest that provides an optimized method for assessment genetic diversity of cultivated Glycyrrhiza uralensis, G. inflate and G. glabra using Chemical fingerprint and ISSR markers which is useful for further investigation in breeding.