Objectives: Osteosarcoma (OS) is one of the aggressive bone tumors commonly diagnosed in adolescents and young adults. The study investigated the effect of 9 single nucleotide polymorphisms (SNPs) in 5 genes on methotrexate (MTX) plasma level and its impact on the patients’ clinical outcomes. Methods: One hundred and thirty-three patients with OS (68 boys and 65 girls with age range 4.00-17.86 years) were enrolled and treated with four weeks of high-dose methotrexate (HDMTX) as neo-adjuvant therapy in children’s cancer hospital (CCHE-57357). Blood samples were collected and genotyped for the studied SNPs (MTHFR rs1801133, SLCO1B1 rs11045879, rs4149081, rs2306283, ABCC3 rs4793665, rs4148412, rs733392 ABCG2 rs2231142, and ABCC2 rs717620 using Taqman® (RT-PCR) assay. Plasma concentrations of MTX were measured using the enzyme multiple immunoassay technique (EMIT®). The clinical outcomes included toxicities that were evaluated based on the common terminology criteria for adverse events - version 5 (CTCAEv5.0.), besides the studying of survival analysis and tumor necrosis (TN %). Results: Older patients experienced increased risk of delayed MTX elimination at 72-hour, OR=1.19 (95% CI=1.09-1.29, p=0.00059). The studied SNPs weren’t associated with MTX elimination. Patients with ABCC2 HW (Homozygous wild) genotype group showed a high statistically significant association with higher grade hyperbilirubinemia, OR= 2.05(95% CI=1.05-4.01, p=0.037). Moreover, the patients with ABCC3 (rs4793665) HV and HT (Homozygous variant and Heterozygous) genotypes had a significant association with severe nephrotoxicity, OR= 0.34(95% CI=1.6-0.72, p=0.005). Conclusions: ABCC2 (rs717620), ABCC3 (rs4793665) genotyping with age and gender could help in predicting patients at risk of toxicities due to HDMTX.