Lipids represent a large family of compounds with highly diverse structures that are involved in complex biological processes. A photocatalytic technique of on-tissue epoxidation of C=C double bonds has been developed for in situ mass spectrometric identification and spatial imaging of positional isomers of lipids. It is based on the plasmonic hot-electron transfer from irradiated gold nanowires to redox-active organic matrix compounds that undergo bond cleavages and generate hydroxyl radicals in nanoseconds. Intermediate radical anions and negative fragment ions have been unambiguously identified. Under the irradiation of a pulsed laser of the third harmonic of Nd3+:YAG (355 nm), the hydroxyl radical-driven epoxidation of unsaturated lipids with different numbers of C=C bonds can be completed in nanoseconds with high yields of up to 95%. Locations of C=C bonds were recognized with diagnostic fragment ions that were produced by either collision with an inert gas or auto-fragmentation resulting from the impact of energetic hot electrons and vibrational excitation. This technique has been applied to the analysis of breast cancer tissues of mice models without extensive sample processes. It was experimentally demonstrated that C=C bonds may be formed at different positions of not only regular mono- or poly-unsaturated fatty acids but also other odd-numbered long-chain fatty acids.