Abstract: Despite persistent questions about the safety of black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa L.), its products continue to be one of the most popular botanical supplements in the United States market. Black cohosh products have been associated with cases of liver toxicity, but subsequent evaluation found some products to be adulterated with other related plants from the same genus. US FDA regulations require that black cohosh products be unadulterated, and correct identification of different species of Actaea is a key first step for their good manufacturing practice. We have developed a phytochemical method to distinguish four different groups of Actaea, including: species other than A. racemosa, Asian species, A. racemosa, and North American species other than A. racemosa. Using HPLC–TOF-ESI-MS technique and principal component analysis, we identified 15 chemical markers (1–3, 5–6, 8–10, 12, 16–21). Three marker compounds were unambiguously identified using authentic standards, and 12 marker compounds were tentatively identified by comparison of fragmentation patterns with previously reported data. The presence of these marker compounds is critical for discrimination among the four groups of closely related plants. The use of metabolic profiling to distinguish black cohosh from related species of Actaea has broader implications in the identification of markers to help authenticate other important medicinal plants. [Copyright &y& Elsevier]