Boosting evidence indicated lipids play important roles in plants. To explore lipid function in cotton fiber development, the lipid composition and content were detected by untargeted and targeted lipidomics. Compared with rapid elongation fibers, the lipid intensity of 16 sub-classes and 56 molecular species decreased, while only 7 sub-classes and 26 molecular species increased in the fibers at the stage of secondary cell wall deposition. Unexpectedly, at the rapid elongation stage, 20 sub-classes and 60 molecular species increased significantly, while only 5 sub-classes and 8 molecular species decreased in the