Methotrexate (MTX), used as a graft-versus-host disease (GvHD) prophylactic agent in hematopoietic stem cell transplantation (HSCT), exerts its effect via folate cycle inhibition. A critical enzyme involved in folate metabolism is 5,10-methylenetetrahydrofolate reductase (MTHFR). We examined the association of a single nucleotide polymorphism (SNP) at position 677 in the MTHFR gene on GvHD outcomes in allogeneic HSCT patients administered MTX. MTHFR genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on 193 HSCT patients and donors. A total of 140 patients were transplanted with an HLA-matched related donor and 53 with an unrelated donor. GvHD outcomes were compared between genotypes by univariate and multivariate analysis. The combined donor 677CT and TT genotypes were associated with a decreased incidence of GvHD (acute and chronic combined) in HSCT recipients with an HLA-matched related donor (75% at 1 year in the CT and TT group compared with 91% in the wild type CC group, P=0.01), increased time to onset of first GvHD (P=0.001) and time to first GvHD treated with systemic therapy (P=0.022). Unrelated donor MTHFR genotype was not associated with outcome parameters and no associations of recipient genotype in either related or unrelated donor cohorts were observed.