Black cohosh [Actaea racemosa L.; syn. Cimicifuga racemosa (L.) Nutt.] is a medicinal plant native to America and the woodlands of eastern North America. The roots and rhizomes of black cohosh, used by Native Americans to ease childbirth and treat menstrual cramps, rheumatism, headaches, coughs, asthma, and snakebites, are currently popular as an herbal remedy in the United States and Europe for the relief of discomfort associated with menopause. To determine chemical variation among populations of this perennial plant, root samples from 33 locations within the natural range of the species, ranging from Massachusetts to South Carolina and west to Missouri and Tennessee, were collected and chemically analyzed by HPLC-PDA using a C-18 reversed phase column (Phenomenex) for separation of the chemical constituents. The constituents were identified by comparison with commercial standards, including actein, methyl-beta-arabinopyranoside, caffeic acid, cimicifugoside, cimicifugoside H1, cimiracemoside A, 26 deoxyactein, ferulic acid, isoferulic acid, kaempferol, formononetin, and others. Chemically, an extract of the root and rhizome is known to contain at least three major natural product groups: cycloartenal-type triterpenes, phenolics, and flavonoids. Chemical constituents, especially formononetin, varied among the populations.