The hypothalamus plays an essential role in the central nervous system of mammals by among others regulating glucose homeostasis, food intake, temperature, and to some extent blood pressure. Assessments of hypothalamic metabolism using, e.g. 1H MRS in mouse models can provide important insights into its function. To date, direct invivo1H MRS measurements of hypothalamus have not been reported. Here, we report that invivo single voxel measurements of mouse hypothalamus are feasible using 1H MRS at 14.1T. Localized 1H MR spectra from hypothalamus were obtained unilaterally (2–2.2 µL, VOI) and bilaterally (4–4.4 µL) with a quality comparable to that of hippocampus (3–3.5 µL). Using LCModel, a neurochemical profile consisting of 21 metabolites was quantified for both hypothalamus and hippocampus with most of the Cramér-Rao lower bounds within 20%. Relative to the hippocampus, the hypothalamus was characterized by high γ-aminobutryric acid and myo-inositol, and low taurine concentrations. When studying transgenic mice with no glucose transporter isoform 8 expressed, small metabolic changes were observed, yet glucose homeostasis was well maintained. We conclude that a specific neurochemical profile of mouse hypothalamus can be measured by 1H MRS which will allow identifying and following metabolic alterations longitudinally in the hypothalamus of genetic modified models.