The biological functions of lipids are entirely dependent on their molecular structures with even small changes in structure─such as different sites of unsaturation─providing critical markers for changes in the underlying metabolism. Conventional mass spectrometry imaging (MSI) approaches, however, face the twin challenges of mixture and structural complexity and are typically unable to differentiate lipid isomers that differ only in the position(s) of carbon-carbon double bonds. Recent coupling of ozone-induced dissociation (OzID) with matrix-assisted laser desorption/ionization (MALDI)-MSI has demonstrated the potential to map changes in individual double-bond isomers, thus enabling visualization of the modulation in lipid desaturation in adjacent tissue types. This has, to date, only been performed in positive-ion mode due to a generally higher abundance of phosphatidylcholines (PC) in mammalian tissues and the efficient desorption/ionization of this lipid subclass. Many other glycerophospholipids (GPLs), however, are better detected in negative-ion mode as deprotonated anions. Recently, OzID has been implemented on a traveling-wave ion-mobility mass spectrometer (Waters, SYNAPT G2-Si) that provides a 50-fold increase in the rate of the gas-phase reaction between ionized lipids and ozone and a commensurate increase in sensitivity for isomer-resolved mass spectrometry. These gains are exploited here to interrogate the distributions of anionic GPL isomers in biological tissues, covering the subclasses phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA). Exploiting both ozone- and collision-induced dissociation in a single acquisition simultaneously identifies sites of unsaturation and acyl chain composition from the same mass spectrum.