Tropical root and tuber crops are staple foods in developing countries where they are the main source of carbohydrates and a potential source of secondary metabolites such as carotenoids. Enhancing carotenoid content has considerable potential for improving human nutrition in these areas. Except for cassava and sweet potato, carotenoid content has not been evaluated in large germplasm collections. We describe a simple HPLC-DAD-based protocol for the reliable characterization of carotenoids in root crops. Less than 5% variation in carotenoid content was found among samples collected from tubers of the same plant or from clones of the same genotype. Carotenoids were evaluated in 153 accessions representing 10 different species. Each species had a specific profile of carotenoids, and accessions of the same species differed greatly in their individual content. The database assembled during this study represents a useful tool for nutritionists and breeders working on biofortification of root crops with carotenoids.