Methotrexate (MTX) is an important drug for the treatment of childhood acute lymphoblastic leukemia(ALL). However, related toxicity occurs in many organs which may cause interruption of treatment, morbidity,and mortality. Single nucleotide polymorphisms (SNPs) of dihydrofolate reductase (DHFR) and gamma glutamylhydrolase (GGH) are known to alter their enzymatic activity and thus affect the metabolism of MTX and influencethe effectiveness. Therefore, we hypothesized that genetic variations of DHFR and GGH genes may influencethe risk of toxicity after high dose MTX. The study population comprised of 105 children with ALL who weretreated according to the modified St Jude Total XV protocol. The patients received 2.5 or 5 g/m2 of MTX for 5doses during the consolidation phase. Genotyping of DHFR 829C>T and GGH -401C>T was performed usinga polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP). The GGH-401CT andTT genotypes were associated with increased risk of leukopenia and thrombocytopenia after high dose MTX(OR 2.97, 95%CI; 1.24-7.13 and OR 4.02, 95%CI; 1.58-10.26). DHFR 829C>T was not associated with toxicity.In conclusion, the GGH -401CT and TT genotypes were found to increase the risk of severe leukopenia andthrombocytopenia after exposure to high dose MTX for childhood ALL therapy.