Methotrexate (MTX) has proven efficient in the treatment of a number of malignancies, as well as non-malignant disorders characterized by a rapid cellular growth. Yet some patients might develop resistance, while others could have toxic side effects. MTX achieves its cytotoxicity through the inhibition of folate-dependent enzymes, suggesting that the genes controlling their activity or the levels of folate cofactors can modulate drug efficacy and, thus, the sensitivity of a patient to MTX. Indeed, several studies, conducted mostly in leukemia and rheumatoid arthritis patients, have addressed the potential for tailoring MTX therapy based on a patient's genetics. Several genetic variants have been shown to have a predictive role, among which the most frequently studied are those of methylenetetrahydrofolate reductase and thymidylate synthase genes. The other candidates, as well as gene-gene interactions, which may be even more important for the prediction of disease outcomes than the individual gene effects, are also briefly discussed.