Analysis of the polar lipids of Toxoplasma gondii by electrospray ionization tandem mass spectrometry provides a detailed picture of the lipid molecular species of this parasitic protozoan. Most notably, T. gondii contains a relatively high level, estimated to about 2% of the total polar lipid, of ceramide phosphoethanolamine. The ceramide phosphoethanolamine has a fatty amide profile with only 16- and 18-carbon species. Compared with the host fibroblasts in which it was grown, T. gondii also has higher levels of phosphatidylcholine, but lower levels of sphingomyelin and phosphatidylserine. Analysis at the molecular species level indicated that T. gondii has greater amounts of shorter-chain fatty acid in its polar lipid molecular species than the host fibroblasts. Shorter-chain fatty acids with a combined total of 30 or fewer acyl carbons make up 21% of Toxoplasma’s, but only 3% of the host’s, diacyl phosphatidylcholine. Furthermore, diacyl phosphatidylcholine with two saturated acyl chains with 12, 14, or 16 carbons make up over 11% of parasite phosphatidylcholine, but less than 3% of the host phosphatidylcholine molecular species. The distinctive T. gondii tachyzoite lipid profile may be particularly suited to the function of parasitic membranes and the interaction of the parasite with the host cell and the host’s immune system. Combined with T. gondii genomic data, these lipidomic data will assist in elucidation of metabolic pathways for lipid biosynthesis in this important human pathogen.