Food loss and waste occurs throughout the agri-food system, manly in the fruit and vegetable and grain sectors, with million tons of residues and by-products generated annually worldwide. Although there is waste considered inevitable, such as potato and citrus peels and watermelon seeds, among many others, this biomass can be upcycled as valuable ingredients of food and biotechnological interest. For example, red-fleshed watermelon contains a large amount of seeds that, despite having high levels of protein and fat, among other constituents, are usually thrown away. In contrast, there is a lack of information in the literature on the composition of yellow-fleshed watermelon seeds. Therefore, this study aimed to evaluate the levels of crude fat, fatty acids, and tocopherols of yellow-fleshed watermelon seeds. Seeds were collected from fruits grown in Bahia, Brazil, washed in tap water, dried at 40°C for 48 h, and ground to a fine powder. Crude fat was determined by Soxhlet extraction and used to characterize the fatty acid profile by gas chromatography with flame-ionization detection, after a derivatization process. Tocopherols were analyzed in a high-performance liquid chromatography system coupled to a fluorescence detector, using tocol as internal standard. It was found that the seed sample contained 17.9 g/100 g of fat and 13.7 mg/100 g of tocopherols, values that do not differ much from those previously reported for red-fleshed watermelon seeds [1]. Seventeen fatty acids were identified and linoleic acid was the most abundant, representing about 77% of the lipid fraction, followed by palmitic and oleic acids. The profile was thus dominated by an essential omega-6 polyunsaturated fatty acid. These results indicate that yellow-fleshed watermelon seeds have high levels of healthy fat and tocopherols (vitamin E), nutrients with several benefits to human health. These seeds can be upcycled as an alternative cheap source of these compounds.