Background: The current study was aimed to elucidate the association of thymidylate synthase (TYMS) 5’-UTR 28bp tandem repeat and cytosolic serine hydroxymethyltransferase (cSHMT) C1420T polymorphisms withacute leukemia in South Indian subjects. A total of 812 subjects [523 healthy controls, 148 acute lymphoblasticleukemia (ALL) cases and 141 acute myeloid leukemia (AML) cases] were screened for TYMS 5’-UTR 28bptandem repeat and cSHMT C1420T using PCR-AFLP and PCR-with confronting two-pair primers (CTPP)approaches. TYMS 5’-UTR 2R allele frequencies of controls, ALL and AML cases were 35.3%, 28.0% and30.1% respectively. This polymorphism conferred protection against ALL (OR: 0.71, 95%CI: 0.53-0.96) whileshowing no statistically significant association with AML (OR: 0.79, 95%CI: 0.58, 1.07). The cSHMT variantallele (T-) frequencies of ALL and AML cases (6.42% and 5.68% respectively) were significantly lower comparedto controls (58.3%). This polymorphism conferred protection against ALL (OR: 0.049, 95%CI: 0.029-0.081)and AML (OR: 0.043, 95%CI: 0.025-0.074). The TYMS 5’-UTR 2R2R genotype was associated with a lowertotal leukocyte count, smaller percentage of blasts, and more adequate platelet count compared to 2R3R and3R3R genotypes in ALL cases. No such genotype-dependent differences were observed in AML cases. ALL casescarrying the cSHMT C1420T polymorphism showed higher disease free survival compared to those with thewild genotype. To conclude, the TYMS 5’-UTR 28bp tandem repeat reduces risk for ALL while cSHMT C1420Treduces risk for both ALL and AML. Both also influence disease progression in ALL.