Meibomian gland dysfunction (MGD) is a leading cause of dry eye disease, yet the underlying lipid metabolic alterations remain poorly understood due to the limited lipid content of meibomian secretions. In this study, we employed LC-MS-based non-targeted lipidomics to comprehensively profile the lipidome of meibum samples from 670 participants, including non-MGD controls and patients with mild, moderate, and severe MGD. Our analysis identified 4,235 lipids across 40 subclasses, revealing significant shifts in lipid composition with disease progression—most notably, increases in wax esters and ceramides, and decreases in diacylglycerols and hexosylceramides.