Objective To evaluate the differences in three kinds of meibomian phospholipid composition in healthy subjects and in patients suffering from meibomian gland dysfunction （MGD）. Methods In this prospective study， 22 MGD patients （MGD group） and 30 age and sex matched healthy individuals （control group） were enrolled. One eye of each subject was included in the study. MGD patients underwent sequential examinations as follows： the property of meibomian secretion， tear film break-up time （BUT）， corneal fluorescein staining （CFS）， Schirmer Ⅰ test and lid margin and ocular surface examinations by slit lamp. Meibomian gland secretions were collected using sterile stainless steel curettes in the healthy control group and MGD group after gentle massage of the lid margin with sterile cotton swabs. The meibomian phospholipids， mainly lysophosphatidylcholine， phosphatidylethanolamine， and sphingomyelin， were detected using liquid chromatography/tandem mass spectrometry （LC-MS/MS）， and comparisons were made between the MGD group and the healthy control group. The data from the two groups were compared by an independent-samples t test and a Pearson correlation analysis was used to assess the correlation between the relative composition concentrations of the three kinds of phospholipids with the clinical parameters of the objective signs of MGD. Results The relative concentration level of lysophosphatidylcholine in the MGD group was significantly higher than that in the healthy controls （t=-2.45， P<0.05）， while phosphatidylethanolamine and sphingomyelin levels were lower in the MGD group than in healthy controls （t=16.73， P<0.01 and t=18.59， P<0.01， respectively）. The relative concentration of lysophosphatidylcholine was positively correlated with the property of meibomian secretion， CFS， lid margin and ocular surface examination by slit lamp （r=0.602， 0.716， 0.710， P<0.01， respectively）； while a negative correlation was found with the BUT and Schirmer Ⅰ test （r=-0.628， -0.477， P<0.05， respectively）. The relative concentrations of phosphatidylethanolamine and sphingomyelin were negatively correlated with the properties of the meibomian secretion， CFS， lid margin and ocular surface examination （r=-0.565， -0.724， -0.702； r=-0.597， -0.719， -0.698， P<0.01， respectively）. However， a positive correlation was detected with the BUT and Schirmer Ⅰ test （r=0.659， 0.498， P<0.05； r=0.631， 0.468， P<0.05， respectively）. Conclusion The profile of the three kinds of meibomian phospholipids （lysophosphatidylcholine， phosphatidylethanolamine and sphingomyelin） in MGD patients is dramatically different from healthy subjects. Its significant correlation with the clinical parameters of the signs of meibomian gland dysfunction suggest that a change in phospholipids composition in meibomian gland secretions is a probable reason for the development of MGD.