The best endoscopic diagnostic strategy for gastrointestinal (GI) graft-versus-host disease (GVHD) is unknown. Over a 48-month period, all patients with unexplained diarrhea at risk for acute gastrointestinal GVHD were prospectively identified. Acute GVHD was defined as symptoms and histologic evidence of GVHD occurring within 100 days of transplant or donor lymphocyte infusion (DLI). Colonoscopy was performed with multiple biopsies of the ileum, right colon and rectosigmoid colon. Next, upper endoscopy with duodenal and random gastric biopsies of both antrum and body were performed. All biopsies were evaluated for GVHD by an experienced GI pathologist. Over the study period, 24 patients (mean age 37 years; 62.5% male) were evaluated. The median time from transplantation or DLI was 30.5 days. The biopsy site with the highest yield was the distal colon (82%). A combination of upper endoscopy with sigmoidoscopy and colonoscopy with ileal biopsies were equivalent (∼94%). In patients with diarrhea at risk for GVHD, biopsies of the distal colon had the highest diagnostic yield suggesting the importance of sigmoidoscopy and biopsy. Colonoscopy and ileoscopy or flexible sigmoidoscopy plus upper endoscopy had the highest diagnostic yields. © 2006 Nature Publishing Group.