Sorghum, a globally grown gluten-free cereal, is used mainly as an animal feed in developed countries regardless of its potential for human consumption. In this study, we utilized nontargeted lipidomics to thoroughly analyze, compare, and characterize whole-grain lipids in six sorghum cultivars (cv) grown in a single field trial in Australia: Buster, Bazley, Cracker, Liberty, MR43, and Tiger. In total, 194 lipid molecular species representing five major lipid classes were identified. Multivariate analysis unveiled distinct lipid profiles among the cultivars. The most distinct lipid profile belonged to cv. MR43. The lower ω-6 to ω-3 ratio and optimal P/S ratio in cv. Bazley reflect this as a valuable source of balanced essential fatty acids in the diet. The novel bioactive lipids known as FAHFAs (fatty acid esters of hydroxy fatty acids) were identified and characterized in sorghum grains. These findings further emphasize the potential of whole-grain sorghum as a basis for new health-promoting food products.