Algae lipids contain long-chain saturated and polyunsaturated fatty acids. The lipids may be transesterified to generate biodiesel fuel. Here, we compare polar lipid compositions for two microalgae, Nannochloropsis oculata and Haematococcus pluvialis, that are prospective lipid-rich feedstock candidates for an emerging biodiesel industry. Online nano liquid chromatography coupled with negative electrospray ionization 14.5 T Fourier transform ion cyclotron resonance mass spectrometry (()ESI FT-ICR MS) with newly modified ion optics provides ultrahigh mass accuracy and resolving power to identify hundreds of unique elemental compositions. Assignments are confirmed by isotopic fine structure for a polar lipid extract. Collision-induced-dissociation (CID) MS/MS provides additional structural information. H. pluvialis exhibits more highly polyunsaturated lipids than does N. oculata.