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Neuronal membrane phospholipids are highly affected by oxidative stress caused by ischemic injury. Thus, it is necessary to identify key lipid components that show changes during ischemia to develop an effective approach to prevent brain damage from ischemic injury. The recent development of MALDI imaging MS (MALDI IMS) makes it possible to identify phospholipids that change between damaged and normal regions directly from tissues. In this study, we conducted IMS on rat brains damaged by ischemic injury and detected various phospholipids that showed unique distributions between normal and damaged areas of the brain. Among them, we confirmed changes in phospholipids such as lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin by MALDI IMS followed by MS/MS analysis. These lipids were present in high concentrations in the brain and are important for maintenance of cellular structure as well as production of second messengers for cellular signal transduction. Our results emphasize the identification of phospholipid markers for ischemic injury and successfully identified several distinctly located phospholipids in ischemic brain tissue.