Current methods to identify, classify, and predict tumor behavior mostly rely on histology, immunohistochemistry, and molecular determinants. However, better predictive markers are required for tumor diagnosis and evaluation. Due, in part, to recent technological advancements, metabolomics and lipid biomarkers have become a promising area in cancer research. Therefore, there is a necessity for novel and complementary techniques to identify and visualize these molecular markers within tumors and surrounding tissue. Since its introduction, mass spectrometry imaging (MSI) has proven to be a powerful tool for mapping analytes in biological tissues. By adding the label-free specificity of mass spectrometry to the detailed spatial information of traditional histology, hundreds of lipids can be imaged simultaneously within a tumor. MSI provides highly detailed lipid maps for comparing intra-tumor, tumor margin, and healthy regions to identify biomarkers, patterns of disease, and potential therapeutic targets. In this manuscript, recent advancement in sample preparation and MSI technologies are discussed with special emphasis on cancer lipid research to identify tumor biomarkers. MSI offers a unique approach for biomolecular characterization of tumor tissues and provides valuable complementary information to histology for lipid biomarker discovery and tumor classification in clinical and research cancer applications.