Background: Osteosarcoma (OS) is one of the aggressive bone tumours commonly diagnosed in adolescents and young adults. Aim: The study aimed to investigate the effect of 9 single nucleotide polymorphisms (SNPs) in 5 genes on methotrexate (MTX) plasma level and the association between the SNPs and MTX toxicities. Subjects and Methods: One hundred and thirty-three children with OS were enrolled and treated with four weeks of high-dose methotrexate (HDMTX) as neo-adjuvant therapy in a children__ampersandsignrsquo;s cancer hospital. Blood samples were genotyped for the studied SNPs (MTHFR rs1801133C__ampersandsigngt;T, SLCO1B1 rs11045879C__ampersandsigngt;T, rs4149081 A__ampersandsigngt;G, rs2306283 A__ampersandsigngt;G, ABCC3 rs4793665C__ampersandsigngt;T, rs4148412T__ampersandsigngt;C, rs733392G__ampersandsigngt;A, ABCG2 rs2231142 G__ampersandsigngt;T, and ABCC2 rs717620C__ampersandsigngt;T using TaqMan__ampersandsignreg; RT-PCR assay. Plasma concentrations of MTX were measured. Toxicities were evaluated, besides the studying of survival analysis and tumour necrosis (TN%). Significance was considered at p __ampersandsignlt; 0.004. Results: Older patients experienced an increased risk of delayed MTX elimination at 72-hour [p = 0.00059, OR 1.19, 95% CI (1.09-1.29)]. Moreover, older patients were associated with protection of developing higher grades of enzymatic hepatotoxicity for (ALT) [p = 0.002, OR 0.88, 95% CI (0.8-0.95)] and (AST) [p = 0.001, OR 0.82, 95% CI (0.76-0.89)]. Patients with (CC) genotypegroup of ABCC3 (rs4793665) C__ampersandsigngt;T(14% of the studied population) associated with protective effect against severe nephrotoxicity [p = 0.003, OR 0.31, 95% CI (0.14-0.67)]. Conclusion: The genotyping of ABCC3 (rs4793665 C__ampersandsigngt;T) and age could help in predicting patients with OS at risk of toxicities due to HDMTX.