Histopathological assessment of lymph node metastases (LNM) depends on subjective analysis of cellular morphology with inter-/intra-observer variability. In this study, LNM from esophageal adenocarcinoma was objectively detected using desorption electrospray ionization-mass spectrometry imaging (DESI-MSI). Ninety lymph nodes and their primary tumor biopsies from 11 esophago-gastrectomy specimens were examined and analyzed by DESI-MSI. Images from mass spectrometry and corresponding histology were co-registered and analyzed using multivariate statistical tools. The MSIs revealed consistent lipidomic profiles of individual tissue types found within lymph nodes. Spatial mapping of the profiles showed identical distribution patterns as per the tissue types in matched immunohistochemistry images. Lipidomic profile comparisons of LNM versus the primary tumor revealed a close association in contrast to benign lymph node tissue types. This similarity was used for the objective prediction of LNM in mass spectrometry images utilizing the average lipidomic profile of esophageal adenocarcinoma. The multivariate statistical algorithm developed for LNM identification demonstrated a sensitivity, specificity, positive predictive value and negative predictive value of 89.5, 100, 100 and 97.2 per-cent, respectively, when compared to gold-standard immunohistochemistry. DESI-MSI has the potential to be a diagnostic tool for peri-operative identification of LNM and compares favorably with techniques currently used by histopathology experts.