We report the first application of online LC-MS (liquid chromatography-mass spectrometry) characterization of algae polar lipids by nanoscale high-performance liquid chromatography followed by electrospray ionization and mass analysis with a linear ion trap (LTQ) coupled with 14.5 T Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ultrahigh FT-ICR mass resolution provides highly accurate mass measurement and resolves monoisotopic peaks from interfering components for unique determination of lipid elemental compositions. We establish the polar lipid profile of fatty acids, glycolipids, phospholipids, and betaine lipids for a green algae, Nannochloropsis oculata, which is highly prized for its oils suitable for biodiesel production. Lipid headgroup and fatty acid identification is based on accurate mass measured by the FT-ICR MS and collision-induced dissociation (CID) MS/MS in the LTQ, Unequivocal lipid composition is further confirmed from isotopic fine structure at baseline resolution-achievable only with ultrahigh resolution FT-ICR MS.