Phospholipids are the main constituent of cell membranes and play multiple roles in cells, in forming the permeability barrier, supporting matrix, surfaces for many catalytic processes, and precursors of signal processing and macromolecular synthesis. Hydrophilic interaction (HILIC) ultra-performance liquid chromatography (UPLC) coupled with quadrupole time of flight (Q-TOF) mass spectrometry (MS) was used to analyze the composition of phospholipids from Schizochytrium sp. S31. Phosphatidyl-cholines, -ethanolamines, -inositoles, and -glycerol, and phosphatidic acid were separated by using an acetonitrile/ammonium formate gradient mobile phase. The structures of 70 phospholipids were identified by LC-MS/MS measurements in negative ion mode. The main phospholipid in Schizochytrium sp. S31, phosphatidylcholine, accounted for 49.8% of the total phospholipids, and the major phospholipid fatty acids were C16 : 0 and DHA.