Comprehensive structural characterization of phosphatidylcholines (PCs) is essential to understanding their biological functions and roles in metabolism. Electron induced dissociation (EID) of protonated PCs directly generated from biological tissues has previously been shown to provide in-depth structural information on the lipid headgroup, regiosiomerism of fatty acyl tails and double bond positions. Although phosphatidylcholine ions formed via alkali metal cationization ( i.e., [M + Na] + and [M + K] + ) are commonly generated during matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry experiments, the gas-phase ion chemistry behavior of EID on sodium- and potassium-cationized phosphatidylcholine ion types has not been studied for ions generated directly from tissue. Herein, we demonstrate EID on [M + Na] + and [M + K] + ion types in a MALDI imaging mass spectrometry workflow for lipid structural characterization. Briefly, near-complete structural information can be obtained upon EID of sodium- and potassium-cationized PCs, including diagnostic fragmentation of the lipid headgroup as well as identification of fatty acyl chain positions and double bond position. EID of cationized lipids generates s n -specific glycerol backbone cleavages as well as a favorable combined loss of sn -2 fatty acid with choline over sn -1, allowing for facile differentiation and relative quantification of PC regioisomers. Moreover, relative quantification of sn- positional isomers from biological tissue reveals that the relative percentages of sodium- and potassium-cationized sn -positional isomers varies significantly in different regions of rat brain tissue.