Methotrexate (MTX) during maintenance therapy is essential to cure acute lymphoblastic leukemia (ALL), but dosing strategies to achieve adequate treatment intensity are challenged by inter-individual differences in drug disposition. To evaluate genetic factors associated with MTX metabolism, we performed a genome-wide association study in 447 ALL cases from the Nordic Society for Paediatric Haematology and Oncology ALL2008 study, validating results in an independent set of 196 patients. The intergenic SNP rs1382539 located in a regulatory element of DHFR was associated with increased levels of short-chain MTX polyglutamates (P = 1.1 × 10-8) due to suppression of enhancer activity, while rs35789560 in FPGS (p.R466C, P = 5.6 × 10-9) was associated with decreased levels of long-chain MTX polyglutamates through reduced catalytic activity. Furthermore, the FPGS variant was linked with increased relapse risk (P = 0.044). These findings show a genetic basis for inter-patient variability in MTX response and could be used to improve future dosing algorithms.