Regimen-related toxicity, one of the main reasons for discontinuation of chemotherapy in acute lymphoblastic leukemia (ALL), can not only be life-threatening but may affect the risk of relapse. In 246 children with newly-diagnosed ALL who were treated using a single protocol (St. Jude Total XIIIB), we used a candidate-gene approach to determine whether acute toxicities [gastrointestinal (stomatitis or diarrhea), hyperbilirubinemia, infection, or neurotoxicity] were related to 16 common polymorphisms in genes plausibly linked to the pharmacodynamics of the drug therapy. During the high-dose methotrexate consolidation therapy, only genotypes related to methotrexate disposition were associated with grade 3–4 gastrointestinal toxicity, adjusting for demographic factors and ALL risk group. Patients having the RFC AA/AG (p = 0.025) genotypes or those having the MTHFR 677 TT (p = 0.048) genotypes had a higher risk of the toxicity (OR = 10.4 and 3.2 respectively), than those with RFC GG or MTHFR CT/CC. In addition, the risk of hyperbilirubinemia during consolidation was higher among those with the lower activity UGT1A1 7/7 genotype than those with other UGT1A1 genotypes (OR = 12.2, p < 0.0001). Adjusting for time at risk over the entire therapeutic period, RFC AA/AG was again prognostic for gastrointestinal toxicity (p < 0.0001); the GSTP1 AA/AG genotypes (p = 0.021) and TPMT heterozygous genotypes (p = 0.047) were also risk factors. The RFC AA/AG genotype was also a modest risk factor for infection (p = 0.05). There were only weak predictors of neurotoxicity, with the MDR1 exon 26 TT genotype a risk factor (p = 0.025) for presumed methotrexate-related neurotoxicity over the entire therapeutic period. We conclude that germline polymorphisms influence the toxicity of antileukemic therapy and their identification may provide a tool for tailoring therapy in childhood ALL.