Significance Tumor cells reprogram their metabolism to support cell growth, proliferation, and differentiation, thus driving cancer progression. Profiling of the metabolic signatures in heterogeneous tumors facilitates the understanding of tumor metabolism and introduces potential metabolic vulnerabilities that might be targeted therapeutically. We proposed a spatially resolved metabolomics method for high-throughput discovery of tumor-associated metabolite and enzyme alterations using ambient mass spectrometry imaging. Metabolic pathway-related metabolites and metabolic enzymes that are associated with tumor metabolism were efficiently discovered and visualized in heterogeneous esophageal cancer tissues. Spatially resolved metabolic alterations hold the key to defining the dependencies of metabolism that are most limiting for cancer growth and exploring metabolic targeted strategies for better cancer treatment.