Gastrointestinal graft-versus-host disease (GVHD) is a common and potentially life-threatening complication after allogeneic hematopoietic stem-cell transplantation (HSCT). Noninvasive tests for assessment of GVHD activity are desirable but lacking. In the present study, we were able to visualize intestinal GVHD-associated inflammation in an allogeneic murine transplantation model by ¹⁸F-fluorodeoxyglucose positron emission tomography (FDG-PET) in vivo. A predominant localization of intestinal GVHD to the colon was verified by histology and fluorescence reflectance imaging of enhanced green fluorescent protein (EGFP)–expressing donor cells. Colonic infiltration by EGFP⁺ donor lymphocytes matched increased FDG uptake in PET examinations. These preclinical data were prospectively translated into 30 patients with suspected intestinal GVHD beyond 20 days after transplantation. A total of 14 of 17 patients with a diagnostic histology showed significant FDG uptake of the gut, again predominantly in the colon. No increased FDG uptake was detected in 13 patients without histologic evidence of intestinal GVHD. Our findings indicate that FDG-PET is a sensitive and specific noninvasive imaging technique to assess intestinal GVHD, map its localization, and predict and monitor treatment responsiveness. Novel targeted tracers for PET may provide new insights into the pathophysiology of GVHD and bear the potential to further improve GVHD diagnosis.