Pre-adult density-associated alterations in the composition of storage lipids may affect the cell membrane fatty acid profile (mainly phospholipids), membrane integrity, and cell function. The present study evaluated the impact of pre-adult density conditions, sex, and the selection regime on the composition of phospholipid fatty acids and lifespan of Drosophila melanogaster. The phospholipid profile of adult flies developed under larval crowding contained a higher proportion of polyunsaturated fatty acids, lower proportion of monounsaturated fatty acids, and greater risk of peroxidation. There was also a negative correlation between the peroxidation index (PI) and longevity. The longevity-selected females showed a lower PI compared with control lines under both densities. The present results indicate that pre-adult density may play a significant role in the lifespan of adult flies by altering the composition of phospholipids and shaping cell membrane bilayers with different susceptibilities to peroxidation.