Tumor necrosis factor (TNF)-alpha plays a significant role in conditioning related toxicities and the development of acute graft-versus-host disease (aGVHD). TNF-alpha gene polymorphisms are associated with rejection after organ transplantation and aGVHD in matched related donor blood and marrow transplantation (BMT) recipients. Few studies have been published on unrelated donor BMT in the pediatric age group. In this study, we examined the relationship between specific polymorphisms in TNF pathway genes and the occurrence and severity of aGVHD. Recipient single-nucleotide polymorphisms (SNPs) in TNF-alpha and TNF receptor superfamily members 1A (TNFRSF1A) and 1B (TNFRSF1B) were investigated. In a multi-institutional Pediatric Blood and Marrow Transplant Consortium trial, a total of 180 pediatric patients (mean age, 11.0 years) were prospectively evaluated for clinical outcomes after matched unrelated donor BMT. All patients received myeloablative conditioning and two-drug GVHD prophylaxis with cyclosporine or tacrolimus, with methotrexate in the majority of patients. TNF-alpha genotypes were not correlated with the overall incidence of aGVHD. Significant associations were seen between TNF-alpha variant alleles and the severity of aGVHD (grade II-IV and grade III-IV), especially when analyzed in whites only (n = 165). Grade II-IV aGVHD was correlated with recipient -857T allele (hazard ratio [HR], 0.47; P = .04), -238A allele (HR, 1.76; P = .002), and d3/d3 genotype (HR, 0.64; P = .03). Severe (grade III-IV) aGVHD was associated with TNF-alpha -1031C allele (HR, 2.38; P = .03), -863A allele (HR, 3.18; P = .003), and d4/d4 genotype (HR, 2.82; P = .01). After adjusting for clinical factors, the association of -1031C, -863A, -238A, and d4/d4 genotypes with severity of aGVHD remained statistically significant. No correlation between selected SNPs in TNFRSF1A or TNFRSF1B and the incidence or severity of aGVHD was found. Our findings indicate clinically important relationships between genetic polymorphisms in TNF-alpha and the severity of aGVHD in this cohort. Improved understanding of this relationship may allow for a risk-adjusted approach to GVHD prevention in pediatric BMT.