It is important to study the regulation mechanism of cotton fiber development. As an important component of the membrane, sphingolipids play an important role in fiber development. We characterized the content and composition of sphingolipids in fiber cells at four developmental stages using UHPLC–MS/MS. We analyzed the differences between the sphingolipid molecule species during the elongation stage and the SCW deposition stage of fiber cells and clarify their function in fiber development.