Radix astragali (root of Astragalus membranaceus) is an important traditional Chinese medicine. It has been used as a tonic herb for thousands of years in China. The water extract of the roots has a wide range of immunopotentiating effects and has been proven to be efficacious as an adjunct cancer therapy. Authentication of the herbal plant is routinely required for general practice in the field of herbal medicine. To facilitate rapid identification of numerous varieties of Radix astragali that are circulating on the herb markets, a rapid molecular genetic method, named 3' untranslated region (3' UTR) sequence-based amplified polymorphism (UAP), has been developed. A cDNA library was first built from transcripts of an authentic A. membranaceus species. Several cDNA clones specific to A. membranaceus were identified through subtractive hybridization of the A. membranaceus cDNA library with Arabidopsis total cellular RNA. On the basis of these cDNA sequences of the 3' untranslated region (3' UTR) of selected cDNA clones, a Polymerase Chain Reaction (PCR) was performed on genomic DNAs of the dry roots of several putative A. membranaceus. PCR fragment length polymorphism was found between A. membranaceus and its relatives. By using this method, it was possible to differentiate the authentic A. membranaceus root from those putative ones obtained from herbal medicine markets. To the authors' knowledge, this is the first paper applying UAP in the authentication of traditional Chinese medicine plants.