Lung cancer is the leading cause of cancer related death in the United States, with patients from rural Appalachia having drastically higher incidence and mortality rates, driven by largely unknown mechanisms. Identifying molecular features that contribute to this health disparity is a critical step in lung cancer research that could lead to predictive biomarkers and personalized therapy for the Appalachia population. Recently, aberrant glycogen accumulation in lung tumors has been reported to promote lung cancer progression. However, the full clinical implications of dysregulated glycogen metabolism and its role in lung cancer health disparities has yet to be explored. Matrix‐assisted laser desorption/ionization‐mass spectrometry imaging (MALDI‐MSI) is a new and innovative technique that combines mass spectrometry with high resolution imaging, enabling the detection of hundreds of analytes with spatial distribution. We recently developed an advanced method to analyze glycogen content in formalin‐fixed paraffin‐embedded tissues using MALDI‐MSI. With this technique, we analyzed glycogen chain length and glycogen phosphate content from tissue microarrays containing over 100 lung cancer patient samples banked at the University of Kentucky. Our patient cohort included adenocarcinoma and squamous cell carcinoma patients from Appalachian and non‐Appalachian counties, with diverse cytopathological features. We found significant glycogen accumulation and higher glycogen phosphate content in lung tumor tissue compared to match normal lung across all patients. Further, we found glycogen accumulation is markedly high in adenocarcinoma patients compared to other lung cancer subtypes. Interestingly, Appalachian adenocarcinoma patients exhibit increased glycogen accumulation and higher glycogen phosphate content than patients from non‐Appalachian counties. These data suggest differential glycogen accumulation is a unique feature of lung tumors from Appalachian adenocarcinoma patients. Our study highlights the potential clinical applications of MALDI‐MSI for digital pathology, biomarker applications, and reveals molecular features that contribute to the health disparity in Appalachian lung cancer patients that warrant further molecular analysis.