The molecular composition of phosphatidylcholines (PCs) in total lipid extracts was characterized by a combination of multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass spectrometer and MS3 fragmentation on an ion trap mass spectrometer. Precursor ion spectra for 50 acyl anion fragments of fatty acids (fatty acid scanning) acquired in parallel increased the specificity and the dynamic range of the detection of PCs and identified the fatty acid moieties in individual PC species. Subsequent analysis of detected PC peaks by MS3 fragmentation on an ion trap mass spectrometer quantified the relative amount of their positional isomers, thus providing the most detailed and comprehensive characterization of the molecular composition of the pool of PCs at the low-picomole level. The method is vastly simplified, compared with conventional approaches, and does not require preliminary separation of lipid classes or of individual molecular species, enzymatic digestion, or chemical derivatization. The approach was validated by the comparative analysis of the molecular composition of PCs from human red blood cells. In the total lipid extract of Madin-Darby canine kidney II cells, we detected 46 PC species with unique fatty acid composition and demonstrated that the presence of positional isomers almost doubled the total number of individual molecular species.