Acute lymphocytic leukemia (ALL) is the most common pediatric cancer worldwide, and is particularly more common in the Indian population. Hence, research is increasingly examining the factors involved in disease development. In the present study, we examined the effect of MTHFR (5,10-methylenetetrahydrofolate reductase) C677T and A1298C polymorphisms in ALL. Blood samples of 135 children with ALL and 142 matched controls were analysed. The presence of MTHFR C677T and A1298C polymorphisms were screened using polymerase chain reaction-restriction fragment length polymorphism based approaches. The frequency of MTHFR 677CC, 677CT and 677TT genotypes were 37.77%, 57.03% and 5.18% in cases and 55.63%, 40.84% and 3.52% in controls, respectively. Frequencies of MTHFR 1298AA, 1298AC and 1298CC genotypes were 30.37%, 61.48% and 8.14% in cases and 45.77%, 47.88% and 6.33% in controls, respectively. The present study inidcates that there is an association between MTHFR gene polymorphisms and ALL. MTHFR variants also showed a gender bias. The frequencies of MTHFR 677CC and 677CT genotypes were 33.33% and 65.51% in males and 45.83% and 41.66% in females. Frequencies of MTHFR 1298AA, 1298AC and 1298CC genotypes were 26.43%, 67.81% and 5.74% in males and 37.5%, 50.0% and 12.5% in females, respectively. It is evident that the male children were more susceptible to ALL compared to female children. Associations found in these studies were significant with respect to gender bias; hence, it is possible that MTHFR C677T and A1298C can be good markers for ALL. Moreover, the possibility also exists that these variants may be influenced by the folate uptake of mothers during pregnancy, thereby influencing the enzyme activity and the ethnicity of the cases examined to date. The gender bias of MTHFR polymorphism in ALL is reported for the first time.