Biotransformation of glucose in organs includes multiple pathways, while quantitative evaluation of percentages of its utilization for individual pathways and their spatial heterogeneity in vivo remain unknown. Imaging MS (IMS) and metabolomics combined with a focused microwave irradiation for rapidly fixing tissue metabolism allowed us to quantify and visualize metabolic fluxes of glucose-derived metabolites in the mouse brain in vivo. At 15 min after the intraperitoneal injection of 13C6-labeled glucose, the mouse brain was exposed to focused microwave irradiation, which can stop brain metabolism within 1 s. Quantification of metabolic intermediates containing 13C atoms revealed that a majority of the 13C6-glucose was diverted into syntheses of glutamate, lactate, and uridine diphosphate (UDP)-glucose. IMS showed that regions rich in glutaminergic neurons exhibited a large signal of 13C2-labeled glutamate. On the other hand, the midbrain region was enriched with an intensive 13C6-labeled UDP-glucose signal, suggesting an active glycogen synthesis. Collectively, application of the current method makes it possible to examine the fluxes of glucose metabolism in a region-specific manner.