Acute graft-versus-host disease (aGVHD) can be triggered by inflammatory conditions, including infections and mucositis. We investigated the association between PCR positivity for gastrointestinal (GI) viruses in stool before hematopoietic cell transplantation (HCT) and intestinal aGVHD using Cox proportional hazard models. We included 48 consecutive HCT patients (28 with malignancies and 20 with nonmalignancies) without GI symptoms before HCT. Fifteen patients were GI virus positive: 9 adenovirus, 3 norovirus, 2 parechovirus, and 1 astrovirus. Overall survival was 58%±8%. The cumulative incidence of aGVHD grade 2 to 4 was 43%±8% (n=18) after a median of 47days (range, 14 to 140). In univariate analysis, GI virus PCR positivity was the only predictor for aGVHD (P=008): within the group of GI virus PCR-positive patients, the cumulative incidence of aGVHD 2 to 4 was 70%±12% versus 29±8% in the PCR-negative group (P=004). In conclusion, GI virus PCR positivity before HCT predicted development of intestinal aGVHD. These results may ultimately affect monitoring, aGVHD prophylaxis, and treatment, as well as rescheduling of elective HCTs.