Black cohosh has become one of the most important herbal products in the US dietary supplements market. It is manufactured from roots and rhizomes of Cimicifuga racemosa (Ranunculaceae). Botanical identification of the raw starting material is a key step in the quality control of black cohosh preparations. The present report summarizes a fingerprinting approach based on high performance liquid chromatography-photodiode array/mass spectrometric/evaporative light scattering detection (HPLC-PDA/MS/ELSD) that has been developed and validated using a total of 10 Cimicifuga species. These include three North American species, Cimicifuga racemosa, Cimicifuga americana, Cimicifuga rubifolia, and seven Asian species, Cimicifuga acerina, Cimicifuga biternat, Cimicifuga dahurica, Cimicifuga heracleifolia, Cimicifuga japonica, Cimicifuga foetida, and Cimicifuga simplex. The chemotaxonomic distinctiveness of the HPLC fingerprints allows identification of all 10 Cimicifuga species. The triterpene glycoside cimigenol-3-O-arabinoside (3), cimifugin (12), and cimifugin-3-O-glucoside (18) were determined to be suitable species-specific markers for the distinction of C. racemosa from the other Cimicifuga species. In addition to identification, the fingerprint method provided insight into chemical interconversion processes occurring between the diverse triterpene glycosides contained in black cohosh. The reported method has proven its usefulness in the botanical standardization and quality control of black cohosh products.