© 2019, The Author(s). Treatment for medulloblastoma (MB) — the most common malignant pediatric brain tumor — includes prophylactic radiation administered to the entire brain and spine due to the high incidence of metastasis to the central nervous system. However, the majority of long-term survivors are left with permanent and debilitating neurocognitive impairments as a result of this therapy, while the remaining 30–40% of patients relapse with terminal metastatic disease. Development of more effective targeted therapies has been hindered by our lack of understanding of the underlying mechanisms regulating the metastatic process in this disease. To understand the mechanism by which MB metastasis occurs, three-dimensional matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) experiments were performed on whole brains from a mouse model of human medulloblastoma. Analyzing the tumor and surrounding normal brain in its entirety enabled the detection of low abundance, spatially-heterogeneous lipids associated with tumor development. Boundaries of metastasizing and non-metastasizing primary tumors were readily defined, leading to the identification of lipids associated with medulloblastoma metastasis, including phosphatidic acids, phosphatidylethanolamines, phosphatidylserines, and phosphoinositides. These lipids provide a greater insight into the metastatic process and may ultimately lead to the discovery of biomarkers and novel targets for the diagnosis and treatment of metastasizing MB in humans.