Objective To evaluate the associations of serum methotrexate (MTX) concentrations and MTHFR gene polymorphisms with delayed metabolism of high-dose MTX and adverse reactions in children with acute lymphoblastic leukemia (ALL). Methods Children with ALL treated at the Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, between August 2021 and December 2023 were included (n=214). Serum MTX concentrations after the first HD-MTX administration and MTHFR C677T and A1298C polymorphisms were determined. Clinical data were retrospectively analyzed. Results Among 214 children with ALL, the C677T TT genotype had a higher rate of delayed metabolism than the CT genotype, and the CC genotype had a higher rate than the CT genotype. For A1298C, the AC genotype was associated with a higher incidence of grade I or higher neutropenia than the AA genotype. Higher MTX concentrations were closely associated with grade Ⅱ or higher renal injury, gastrointestinal reactions, and hyperbilirubinemia. Intermediate/high-risk disease category, age >14 years, and body mass index ≥17 kg/m² were risk factors for delayed metabolism. Compared with the C677T CC genotype, the CT genotype had a reduced risk of delayed metabolism, whereas no significant difference was observed between TT and CC. Conclusions Serum MTX concentration serves as an objective marker of MTX-related toxicity. Under adequate rescue therapy and concentration monitoring, a single MTHFR polymorphism appears insufficient to guide dose adjustment. A combined strategy is recommended, with concentration monitoring as the primary approach and genetic factors as an adjunct.