The maximal lifespan (MLS) of mammals is inversely correlated with the peroxidation index, a measure of the proportion and level of unsaturation of polyunsaturated fatty acids (PUFA) in membranes. This relationship is likely related to the fact that PUFA are highly susceptible to damage by peroxidation. Previous comparative work has examined membrane composition at the level of fatty acids, and relatively little is known regarding the distribution of PUFA across phospholipid classes or phospholipid molecules. In addition, data for humans is extremely rare in this area. Here we present the first shotgun lipidomics analysis of mitochondrial membranes and the peroxidation index of skeletal muscle, liver, and brain in three mammals that span the range of mammalian longevity. The species compared were mice (MLS of 4 years), pigs (MLS of 27 years), and humans (MLS of 122 years). Mouse mitochondria contained highly unsaturated PUFA in all phospholipid classes. Human mitochondria had lower PUFA content and a lower degree of unsaturation of PUFA. Pig mitochondria shared characteristics of both mice and humans. We found that membrane susceptibility to peroxidation was primarily determined by a limited number of phospholipid molecules that differed between both tissues and species.