Stemona sessilifolia, S. japonica and S. tuberosa are the three original sources of Stemonae Radix specified in the Chinese Pharmacopoeia (CP), and have been traditionally used for antitussive and insecticidal remedy. Significant variations in alkaloids composition and content, as well as different degrees of antitussive activities were found among them. In order to identify the genuine sources of Stemonae Radix accurately in genetic level, we determined the nucleotide sequences of chloroplast DNA trnL-trnF, trnH-psbA, petB-petD and trnK-rps16 regions of the species recorded in CP and S. parviflora, as well as the common counterfeits of Stemonae Radix, Asparagus species. The results revealed that the sequences of petB-petD and trnK-rps16 regions, showing relatively high substitution rate, were more informative than those of trnL-trnF and trnH-psbA regions. The sequences from all the four regions provided useful information to discriminate the three CP species from each other and from S. parviflora and the counterfeits. A phylogenetic tree reconstructed by the trnH-psbA sequences for 9 Stemona species distributed in China and Thailand showed that the three CP species belonged to the same clade, among which S. japonica and S. sessillifolia formed a sister group, showing closer relations to each other than to S. tuberosa. By contrast, S. parviflora was genetically far from the three CP species. Intra-species variations were observed in the three CP species. Especially, in S. tuberosa two types of petB-petD sequence and four types each of trnL-trnF, trnK-rps16 and trnH-psbA sequences resulted in 6 haplotypes; whereas, these differences had no relation with the different chemical types, but seemed to be consistent with geographical distribution.