The increasing analytical speed of mass-spectrometry imaging (MSI) has led to growing interest in the medical field. Acute kidney injury is a severe disease with high morbidity and mortality. No reliable cut-offs are known to estimate the severity of acute kidney injury. Thus, there is a need for new tools to rapidly and accurately assess acute ischemia, which is of clinical importance in intensive care and in kidney transplantation. We investigated the value of MSI to assess acute ischemic kidney tissue in a porcine model. A perfusion model was developed where paired kidneys received warm (severe) or cold (minor) ischemia (n = 8 per group). First, ischemic tissue damage was systematically assessed by two blinded pathologists. Second, MALDI-MSI of kidney tissues was performed to study the spatial distributions and compositions of lipids in the tissues. Histopathological examination revealed no significant difference between kidneys, whereas MALDI-MSI was capable of a detailed discrimination of severe and mild ischemia by differential expression of characteristic lipid-degradation products throughout the tissue within 2 h. In particular, lysolipids, including lysocardiolipins, lysophosphatidylcholines, and lysophosphatidylinositol, were dramatically elevated after severe ischemia. This study demonstrates the significant potential of MSI to differentiate and identify molecular patterns of early ischemic injury in a clinically acceptable time frame. The observed changes highlight the underlying biochemical processes of acute ischemic kidney injury and provide a molecular classification tool that can be deployed in assessment of acute ischemic kidney injury.