Thin-layer chromatography (TLC) is an essential method for food composition analyses such as lipid nutrition analysis. TLC can be used to obtain information about the lipid composition of foods; however, it cannot be used for analyses at the molecular level. Recently we developed a new method that combines matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) with TLC-blotting (TLC-Blot-MALDI-IMS). The combination of MALDI-IMS and TLC blotting enabled detailed and sensitive analyses of lipids. In this study, we applied TLC-Blot-MALDI-IMS for analysis of major phospholipids extracted from bluefin tuna. We showed that TLC-Blot-MALDI-IMS analysis could visualize and identify major phospholipids such as phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin.