Among several theories involved in the pathogenesis of primary open-angle glaucoma (POAG), the vascular theory considers the disease to be a consequence of reduced ocular blood flow associated with red blood cell abnormalities. Red blood cell membrane structure and function are influenced by their phospholipid composition. We investigated whether specific lipid entities that may affect the membrane physiology, namely, polyunsaturated fatty acids (PUFAs) and plasmalogens, are modified in POAG and whether these potential variations are related to the stage of glaucoma. Blood samples were collected from 31 POAG patients and 10 healthy individuals. The stage of glaucoma was determined according to the Hodapp and Parrish classification. Lipids were extracted from red blood cell membranes and individual phospholipid species were quantified by liquid chromatography combined with mass spectrometry using triple quadrupole technology. POAG patients had reduced erythrocyte levels of phosphatidyl-choline (PC) carrying docosahexaenoic acid (DHA). POAG patients also displayed lower levels of choline plasmalogens (PlsC) carrying PUFAs other than DHA. These differences were greater as the severity of the disease increased. Linear regressions predicted that red blood cell PlsC levels would decrease years before clinical symptoms, whereas the levels of PC carrying DHA were linearly correlated to visual field loss. Our data demonstrate the selective loss of some individual phospholipid species in red blood cell membranes, which may partly explain their loss of flexibility in POAG.