Revealing the inherent molecular diversity of lipid biology requires advanced analytical technologies. Distinguishing phospholipids that differ in the position(s) of carbon-carbon double bonds within their acyl chains presents a particular challenge because of their similar chromatographic and mass spectral behaviours. Here—for the first time—we combine reversed-phase liquid chromatography for separation of isomeric phospholipids with on-line mass spectral analysis by ozone-induced dissociation (OzID) for unambiguous double bond position assignment. The customised tandem linear ion-trap mass spectrometer used in our study is capable of acquiring OzID scans on a chromatographic timescale. Resolving the contributions of isomeric lipids that are indistinguishable based on conventional mass spectral analysis is achieved using the combination of liquid chromatography and OzID. Application of this method to the analysis of simple (egg yolk) and more complex (sheep brain) extracts reveals significant populations of the phosphatidylcholine PC 16:0_18:1(n−7) alongside the expected PC 16:0_18:1(n−9) isomer.