Endogenous phosphatidylcholine in biological membranes exists as isomers with acyl moieties at the sn-1 or sn-2 positions of the glycerol backbone. However, detailed biochemical information on these positional isomers is not generally available. This study is the first report on the separation and identification of positional isomers of endogenous phosphatidylcholine using reversed-phase LC-ESIMS/MS. The separation of positional isomers in PC was achieved by using ultra performance LC, which uses a high-resolution HPLC system. To identify positional isomers in individual PC species, their lyso-PC-related fragments and fatty acids, which were obtained by MS/MS analysis in the negative ion mode, were used. From the application results of biological samples, the lipid extracts of mouse brain were found to be abundant in PC containing 22:6 at the sn-1 position of the glycerol backbone. However, the lipid extracts from mouse heart and liver were not abundant in positional isomers. This achievement demonstrates that the relative amounts of positional isomers in various tissues or molecular species differ. These results will be useful for the clarification of the biological mechanisms of remodelling enzymes such as phospholipase and acyltransferase. Thus, our report provides a novel and critical milestone in understanding how molecular composition of phospholipids is established and their biological roles.