Steroidal glycoalkaloids are naturally occurring, secondary plant metabolites that are found in foods, including potatoes and tomatoes. Their content in plants is controlled by both genetic and environmental factors. Glycoalkaloid profiles can be passed to progenies during breeding and hybridization of wild and cultivated potatoes designed to develop improved potatoes. The most common potato, Solanum tuberosum, contains primarily the glycoalkaloids, alpha-solanine and alpha-chaconine. However, wild-type potatoes being used for breeding new varieties contain other, less common glycoalkaloids. Because glycoalkaloid composition is a major criterion for the release of new potato cultivars, we used HPLC, TLC, GC, and GC/MS to determine their nature and content in several Solanum species widely used in potato breeding and hybridization programs. Solanum tuberosum, as well as S. andigena and S. stenotomum, contained alpha-solanine and alpha-chaconine. S. canasense was found to contain only dehydrocommersonine. S. acaule contained alpha-tomatine and demissine. S. juzepczukii and S. curtilobum contained demissine and two previously unidentified glycoalkaloids. We characterized them as demissidine-glucose/rhamnose (1/1 ratio) and demissidine-galactose/glucose/rhamnose (1/1/1 ratio), tentatively named dihydro-beta(1)-chaconine and dihydrosolanine, respectively. We found extensive variability in the glycoalkaloid profiles in the tested potato varieties. The possible significance of these findings for plant breeding and food safety is discussed.