Precise structural identification of photosynthetic polar glycerolipids in microalga Tetraselmis chuii has been established using Ultra Performance Liquid Chromatography-Electrospray ionization-Quadrupole-Time of Flight Mass Spectrometry (UPLC-ESI-Q-TOF-MS) by direct analysis of the total lipids extract. The mass spectrometry was performed in reflective time-of-flight using electron spraying ionization in both positive and negative modes. The structural determination was based on the characteristic product ions yielded by different glycerolipids under ESI-MS/MS mode, and confirmed the molecular species by the carboxylate anions produced by glycerolipids in the negative mode. As a result, more than 40 lipid molecular species, including 11 monogalactosyldiacylglycerols (MGDG), 7 digalactosyldiacylglycerols (DGDG), 16 sulfoquinovosyldiacylglycerols (SQDG), and 9 phosphatidylglycerols (PG), were detected in Tetraselmis chuii, which had never been identified before in this microalga. Furthermore, some intact lipid molecules with hydroxylated fatty acids that could not be detected by the traditional GC-MS method were found this time, providing novel information for the photosynthetic lipidome of Tetraselmis chuii. Comparative studies on fatty acids at the sn-2 position showed that SQDG and MGDG are dominantly biosynthesized through the prokaryotic pathway, PG is a typically mixed biosynthetic pathway, while DGDG is somewhat peculiar with C14:0 and C16:0 at its sn-2 position. This method could provide a full structural profile of intact photosynthetic lipid molecular species, which may be applied to study the physiological and ecological functions of lipid by monitoring their individual changes.