Historically, studies of brain metabolism have been based on targeted analyses of a limited number of metabolites. Here we present an untargeted mass spectrometry-based metabolomic strategy that has successfully uncovered differences in a broad array of metabolites across anatomical regions of the mouse brain. The NSG immunodeficient mouse model was chosen because of its ability to undergo humanization leading to numerous applications in oncology and infectious disease research. Metabolic phenotyping by hydrophilic interaction liquid chromatography and nanostructure imaging mass spectrometry revealed both water-soluble and lipid metabolite patterns across brain regions. Neurochemical differences in metabolic phenotypes were mainly defined by various phospholipids and several intriguing metabolites including carnosine, cholesterol sulfate, lipoamino acids, uric acid, and sialic acid, whose physiological roles in brain metabolism are poorly understood. This study helps define regional homeostasis for the normal mouse brain to give context to the reaction to pathological events.