Sutherlandia frutescens (L.) R.Br. is extensively used in South Africa to treat a broad range of ailments such as the common cold and cancers, and recently, as adjunct therapy in HIV/AIDS. However, scientific studies focusing on the phytochemical profile and variation within and between populations of this ethnomedicinally important species are lacking. The amino acids; GABA, L-canavanine, L-asparagine, L-arginine and a glycan, D-pinitol have been proposed as being responsible for the pharmacological activity ascribed to S. frutescens. Aerial parts of S. frutescens were collected from several natural populations and cultivation sites. All twenty naturally occurring amino acids together with GABA, L-canavanine and D-pinitol were quantified by LC-MS. Amino acids collectively constituted between 10% and 15% (w/w) of dried plant material. Proline, L-asparagine and alanine were the most abundant amino acids identified and collectively represent approximately 60% of total amino acid content; however, not all samples contained all the amino acids. The yields of the various compounds showed tremendous variation both within and between populations. For example, L-canavanine (a non-protein amino acid) ranged from 0.14 to 13.58 mg/g. This is the first report on the variation of phytoconstituents claimed to contribute to the pharmacological properties of Cancer bush. This information could be of value to select specific chemotypes for cultivation and for developing guidelines for quality control purposes.