Visualization of endogenous molecules in small experimental animals is very important in order to investigate the localization of specific molecules. Conventional imaging techniques are not suitable for simultaneous visualization of endogenous molecules. We recently developed a new imaging method-imaging mass spectrometry (IMS) using matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight (MALDI-QIT-TOF). In this study, we applied IMS using MALDI-QIT-TOF to visualize the metabolites in medaka (Oryzias latipes) sections and determined the spatial distribution of metabolites including lipids.