Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Imaging (MALDI-MSI) has developed as a useful tool in generating comprehensive metabolite profiles along with spatial information in model organisms. The ability of MALDI-MSI to generate in situ profiles in whole organisms or specific tissue sections serves as an efficient alternative to solvent extraction protocols, especially for the identification of biomolecules that are unstable under extraction conditions. Herein, we have utilized MALDI-MSI to spatially profile various classes of lipids in two model organisms, Drosophila melanogaster (fruit fly), and Tribolium castaneum (red flour beetle). Five different classes of phospholipids were imaged in positive ionization mode in both insect species. Confirmation of the m/z assignment for selected lipids was performed using on-tissue MS/MS fragmentation.