A lipid layer consisting of meibum lipids exists in the tear film and functions in preventing dry eye disease. Although the meibum lipids include diverse lipid classes, the synthesis pathway and role of each class remain largely unknown. Here, we created single and double knockout (KO and DKO, respectively) mice for the two acyl-CoA wax alcohol acyltransferases ( and ) and investigated their dry eye phenotypes and meibum lipid composition. KO and DKO mice exhibited severe dry eye with meibomian gland dysfunction, whereas KO mice had mild dry eye. In these mice, specific meibum lipid classes were reduced: (-acyl)-ω-hydroxy fatty acids and type 1ω wax diesters in KO mice, wax monoesters and types 1ω and 2ω wax diesters in KO mice, and most of these in DKO mice. Our findings reveal that Awat1 and Awat2 show characteristic substrate specificity and together produce diverse meibum lipids.