Acute graft-versus-host disease (GVHD) of the gastrointestinal (GI) tract is an often lethal complication of allogeneic hematopoietic cell transplant. Clinical severity correlates with outcomes, but histopathologic grading is primarily used to confirm the clinical diagnosis. One barrier to using histopathologic grading to predict clinical outcomes is inter-grader variability among transplant centers. Recent experimental models have shown that the loss of Paneth cells, which are located in the small intestine and help regulate the GI microbiome by secreting antimicrobial peptides, correlates with clinical GVHD severity. Because Paneth cells are easy to identify and quantify by light microscopy, we evaluated the mean number of Paneth cells per high-powered field (hpf) in 116 duodenal biopsies obtained at diagnosis of GI GVHD at 2 different centers with their clinical outcomes. Paneth cell counts were reproducible between centers (r² = 0.81; P < .0001). Lower numbers of Paneth cells at diagnosis correlated with clinically more severe GI GVHD (P < .0001) and less likelihood of response to GVHD treatment (P < .0001). A threshold of 4 Paneth cells per hpf stratified patients according to nonrelapse mortality (28% vs 56%; P = .004). We conclude that the enumeration of duodenal Paneth cells is a readily available index of disease severity that provides important information regarding GVHD prognosis.