Desorption electrospray ionization (DESI) mass spectrometry imaging has become a powerful strategy for analysis of tissue sections, enabling differentiation of normal and diseased tissue based on changes in the lipid profiles. The most common DESI workflow involves collection of MS1 spectra as the DESI spray is rastered over a tissue section. Relying on MS1 spectra inherently limits the ability to differentiate isobaric and isomeric species or evaluate variations in the relative abundances of key isomeric lipids, such as double-bond positional isomers which may distinguish normal and diseased tissues. Here, 193 nm ultraviolet photodissociation (UVPD), a technique capable of differentiating double-bond positional isomers, is coupled with DESI to map differences in the double-bond isomer composition in tissue sections in a fast, high throughput manner compatible with imaging applications.