Studies had shown that genetic polymorphism plays a significant role in the pharmacokinetics and pharmacodynamics variation of high dose methotrexate (MTX), 5000 mg/m2 regimen. The objective of this study was to investigate the genetic variations associated with the serum level and toxicity of MTX in Malaysian children with acute lymphoblastic leukemia (ALL). Thirty-eight patients were genotyped for rs717620 (ABCC2), rs4948496 (ARID5B), rs1801133 (MTHFR) and rs4149056 (SLCO1B1). Serum levels of MTX at 48 h post 24 h of intravenous infusion were analyzed by high-performance liquid chromatography-mass spectrometry. The ABCC2 genotype was significantly associated with the serum levels of MTX at 48 h after treatment (p = 0.017). Patients with CT and TT of rs717620 (ABCC2) and TC and CC of rs4948496 (ARID5B) were significantly associated with leukopenia grade I-IV (Fisher Exact Test; p = 0.03 and 0.02, respectively). The three most common MTX related toxicities were leukopenia (60.5%), increased alanine aminotransferase enzyme (47.4%), and thrombocytopenia (47.4%). Our results demonstrate that by prescreening of patients for ABCC2 and ARID5B associated with the serum levels and adverse effects of MTX would identify patients at risk and therefore help a pediatric oncologist to personalize chemotherapy drugs for precision health.