We previously reported that tumor mitochondrial redox state and its heterogeneity distinguished between the aggressive and the indolent breast cancer xenografts, suggesting novel metabolic indices as biomarkers for predicting tumor metastatic potential. Additionally, we reported that the identified redox biomarkers successfully differentiated between the normal breast tissue and the cancerous breast tissue from breast cancer patients. The aim of the present study was to further characterize intratumor heterogeneity by its distribution of mitochondrial redox state and glucose uptake pattern in tumor xenografts and to further investigate the metabolic heterogeneity of the clinical biopsy samples. We employed the Chance redox scanner, a multi-section cryogenic fluorescence imager to simultaneously image the intratumor heterogeneity in the mitochondrial redox state and glucose uptake at a high spatial resolution (down to 50 × 50 × 20 μm3). The mitochondrial redox state was determined by the ratio of the intrinsic fluorescence signals from reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp including FAD, i.e., flavin adenine dinucleotide), and the glucose uptake was measured using a near-infrared fluorescent glucose-analogue, pyropheophorbide 2-deoxyglucosamide (Pyro-2DG). Significant inter- and intratumor metabolic heterogeneity were observed from our imaging data on various types of breast cancer xenografts. The patterns and degrees of heterogeneity of mitochondrial redox state appeared to relate to tumor size and metastatic potential. The glucose uptake was also heterogeneous and generally higher in tumor peripheries. The oxidized and reduced regions mostly corresponded with the lower and the higher pyro-2DG uptake, respectively. However, there were some regions where the glucose uptake did not correlate with the redox indices. Pronounced glucose uptake and high NADH were observed in certain localized areas within the tumor necrotic regions, indicative of the existence of viable cells which was also supported by the H&E staining. Significant heterogeneity of the redox state indices was also observed in clinical specimens of breast cancer patients. As abnormal metabolism including the Warburg effect (high glycolysis) plays important roles in cancer transformation and progression, our observations that reveal the 3D intratumor metabolic heterogeneity as a characteristic feature of breast tumors are of great importance for understanding cancer biology and developing diagnostic and therapeutic methods.