Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool that has allowed researchers to directly probe tissue molecular structure and drug content with minimal manipulations, while maintaining anatomical integrity. In the present work glycerophospholipids and sphingolipids images were acquired from 16 µm thick coronal rat brain sections using MALDI-MS. Images of phosphatidylinositol 38:4 (PI 38:4), suifatide 24:1 (ST 24:1), and hydroxyl sulfatide 24:1 (ST 24:1 (OH)) were acquired in negative ion mode, while the images of phosphatidylcholine 34:1 (PC 34:1), potassiated phosphatidylcholines 32:0 (PC32:0 + K(+)) and 36:1 (PC 36:1 +K(+)) were acquired in positive ion mode. The images of PI 38:4 and PC 36:1+K(+) show the preferential distribution of these two lipids in gray matter; and the images of two sulfatides and PC 32:0+K(+) show their preferential distribution in white matter. In addition, the gray cortical band and its adjacent anatomical structures were also identified by contrasting their lipid makeup. The resulting images were compared to lipid images acquired by secondary ion mass spectrometry (SIMS). The suitability of TLC sprayers, Collison Nebulizer, and artistic airbrush were also evaluated as means for matrix deposition.