Methotrexate/6-mercaptopurine maintenance therapy improves acute lymphoblastic leukemia (ALL) outcome. Cytotoxicity is mediated by DNA incorporation of thioguanine nucleotides (DNA-TG). We investigated the association of DNA-TG to relapse risk in 1 910 children and young adults with non-high risk ALL. In a cohort-stratified Cox regression analysis adjusted for sex, age, and white cell count at diagnosis, the relapse-specific hazard ratio (HRa) per 100 fmol/μg increase in weighted mean DNA-TG (wmDNA-TG) was 0.87 (95% CI 0.78-0.97; p = 0.013) in the 839 patients who were minimal residual disease (MRD) positive at end of induction therapy (EOI), whereas this was not the case in EOI MRD-negative patients (p = 0.76). Validation analysis excluding the previously published Nordic NOPHO ALL2008 pediatric cohort yielded a HRa of 0.92 (95% CI 0.82-1.03; p = 0.15) per 100 fmol/μg increase in wmDNA-TG in EOI MRD-positive patients. If also excluding the United Kingdom cohort, in which samples were taken non-randomly in selected patients, the HRa for the EOI MRD-positive patients was 0.82 (95% CI 0.68-0.99; p = 0.044) per 100 fmol/μg increase in wmDNA-TG. The importance of DNA-TG as a biomarker for maintenance therapy intensity calls for novel strategies to increase DNA-TG, although its clinical value may vary by protocol backbone.