Methotrexate (MTX), a folate antagonist employed for treating a wide range of malignancies, has an extensive interpatient variability, resulting in unpredictable toxicity. The present study evaluated the impact of single gene polymorphisms (SNPs: rs1801133 and rs1801131 in the MTHFR gene; rs4149056 and rs11045879 in the SLC01B1 gene; and rs2032582 and rs1045642 in the ABCB1 transporter gene) on MTX blood levels and toxicity in samples from 69 patients with diffuse large-B-cell lymphoma (DLBCL) treated with high dose intravenous (HD IV) MTX, > 2 g/m(2). None of the studied genotypes was found to be associated with a statistically significant risk for elevated MTX levels at 24-48 h after completing therapy with MTX. Ancestral alleles (T) for SLC01B1 rs4149056 (T521C) and SLC01B1 rs11045879 (intron C21273886T) were found to be associated with an increased risk for MTX-related toxicity (p < 0.05 and p = 0.07, respectively), emphasizing the potential importance of employing pharmacogenetic assessment for personalized medicine.