Background: Primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL) is a relatively rare and aggressive neoplasm. High-dose methotrexate (HD-MTX) is an effective regimen for the treatment of PCNS-DLBCL, but MTX–related toxicity remains a problem. The aim of this analysis study was to investigate the influence of the methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism on HD-MTX–related toxicity in patients with PCNS-DLBCL. Material/Methods: A prospective, observational study was conducted to analyze 148 MTX courses in 32 patients with PCNS-DLBCL. Results: The delayed MTX clearance was observed in 53 cycles (35.8%). The patients carrying the homozygous variant genotype had a higher risk of developing nephrotoxicity than those carrying the wild-type genotype (odds ratio [OR] 13.08; 95% confidence interval [CI], 1.65-103.86; P = .002) or heterozygous variant genotype (OR 8.43; 95% CI, 2.31-30.70; P < .001). Significant differences were observed in hepatotoxicity (OR 9.33; 95% CI, 2.54-34.27; P < .001) and hematologic toxicity (OR 3.09; 95% CI, 1.18-8.07; P = .024) in addition to nephrotoxicity between the homozygous variant genotype and the wild-type genotype. Conclusion: The homozygous mutation of C to T at nucleotide 677 increases the risk on HD-MTX–related toxicity. The MTHFR C677T polymorphism can be used to predict HD-MTX–related toxicity for patients with PCNS-DLBCL. This prospective study investigated the influence of the methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism on high-dose methotrexate (HD-MTX)-related toxicity in patients with rare PCNS-DLBCL. A total of 148 cycles of 32 patients were included. The homozygous mutation at nucleotide 677 increases the risk on HD-MTX–related toxicity and can be used as a predictive marker for patients with PCNS-DLBCL. © 2020 Elsevier Inc.