Double bond position and stereochemistry in unsaturated lipids can have profound impact on biological properties and activities but the assignment of these features by mass spectrometry is frequently challenging. Conventional techniques for lipid identification rely on collision-induced dissociation (CID) and are most often unable to differentiate between lipid isomers, particularly those involving double bond position and geometry (i.e., cis and trans). In this study, CID performed on proton-bound complexes of fatty acid methyl esters and iodoaniline (and related reagents) reveals unusual fragmentation patterns. CID products are shown to result from proton transfer and are associated with specific structures of the unsaturated lipids. Notably, CID of these complexes can not only distinguish cis- and trans-fatty acid methyl esters, but also differentiate conjugated double bond arrangements from non-conjugated analogs. Herein, the mechanisms underpinning this unique CID behavior are investigated by stable isotope labeling and are proposed to involve both carbene and free radical intermediates.