The genus Asparagus represents highly valuable plant species having therapeutic and nutraceutical importance. The plant Asparagus racemosus is one of the most widely used sources of phytoestrogens because of its high content of the steroidal saponins, shatavarins I–IV, in roots. The dry root of A. racemosus, known as “Rak-Sam-Sip” in Thai, is one of the most popular herbal medicines. Recently, the interest in plant-derived estrogens has increased tremendously, making A. racemosus particularly important and a possible target for fraudulent labeling. However, the identification of A. racemosus is generally difficult due to its similar morphology to other Asparagus spp. Thus, accurate authentication of A. racemosus is essential. In this study, 1,557-bp nucleotide sequences of the maturase K (matK) gene of eight Asparagus taxa were analyzed. Ten polymorphic sites of nucleotide substitutions were found within the matK sequences. A. racemosus showed different nucleotide substitutions to the other species. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the matK gene was developed to discriminate A. racemosus from others. Ten commercially crude drugs called “Rak-Sam-Sip” were also analyzed by PCR-RFLP technique. Root extracts from eight Asparagus species were evaluated for antioxidant and cytotoxicity. All extracts exhibited weak antioxidant activity. Interestingly, crude extract of A. setaceus showed significant cytotoxic activity against KB cells and NCI-H187 cells with IC50 of 19.59 and 32.60 µg/ml, respectively. This is the first time that cytotoxicity of A. setaceus has been reported.