Lipids are a structurally diverse class of molecules with important biological functions including cellular signaling and energy storage. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows for direct map-ping of biomolecules in tissue. Fully characterizing the structural diversity of lipids remains a challenge due to the presence of isobaric and isomeric species, which greatly complicates data interpretation when only m/z information is available. Integrating ion mobility separations aids in deconvoluting these complex mixtures and addressing the challenges of lipid IMS. Here we demonstrate that a MALDI quadrupole time-of-flight (Q-TOF) mass spectrometer with trapped ion mobility spectrometry (TIMS) enables approximately a ~270% increase in the peak capacity during IMS experiments. MALDI TIMS-MS separation of lipid isomer standards, including sn-backbone isomers, acyl chain isomers, as well as double bond positional and geometric isomers are demonstrated. As a proof-of-concept, in situ separation and imaging of lipid isomers with distinct spatial distributions was performed using tissue sections from a whole-body mouse pup.