Updated project metadata. Different aspects of intra-tumor heterogeneity (ITH), which are associated with development of cancer and its response to treatment, have postulated prognostic value. Here we searched for the potential association between phenotypic ITH analyzed by mass spectrometry imaging (MSI) and the prognosis of head and neck cancer. The study involved tissue specimens resected from 77 patients with locally advanced oral squamous cell carcinoma, including 37 patients where matched samples of primary tumor and synchronous lymph node metastases were analyzed. A 3-year follow-up was available for all patients that enabled their separation into two groups: with no evidence of disease (NED, n=41) and with progressive disease (PD, n=36). After the on-tissue trypsin digestion, peptide maps of all cancer regions were segmented using an unsupervised approach to reveal their intrinsic heterogeneity. We found that intra-tumor similarity of spectra was higher in the PD group and diversity of clusters identified during image segmentation was higher in the NED group, which indicated a higher level of ITH in patients with more favorable outcomes. Furthermore, signature of molecular components that correlated with long-term outcomes could be associated with proteins involved in the immune functions. Hence, we proposed that a higher level of ITH revealed by MSI in cancers with a better prognosis could reflect the presence of heterotypic components of tumor microenvironment such as infiltrating immune cells enhancing the response to the treatment. LC-MALDI-MS/MS data deposited in this repository originate from protein lysates obtained from FFPE sections consecutive to sections dedicated to MSI measurements. The hypothetical identity of the MSI components was established by assignment of a component location on the m/z scale for the measured masses of tryptic peptides identified by LC-MALDI-MS/MS allowing ±0.05% mass tolerance.