Abstract Introduction. High dose (2 – 3 g/m2) methotrexate (MTX, an anti-folate) is used in the treatment of pediatric acute lymphoblastic leukemia (ALL) with side effects in a considerable number of children. We studied the prevalence of MTX related side effects and a possible relation with common polymorphisms in folate metabolism related genes in the treatment of childhood ALL. Methods. In this retrospective study, DNA was isolated from leukemic samples of 81 children with ALL. High risk (HR) ALL patients were treated with 4 courses of 3 g/m2, non-high risk (NHR) patients with 3 times 2g/m2. The following effects were monitored: serum MTX levels, transfusion need, liver toxicity, skin toxicity and hematological toxicity. Common gene mutations in methylenetetrahydrofolate reductase (MTHFR 677C>T and MTHFR 1298A>C), methionine synthase (MS 2756A>G), methionine synthase reductase (MTRR 66A>G), serine hydroxymethyl transferase (SHMT 1420C>T), methylenetetrahydrofolate dehydrogenase (MTHFD1 1958G>A), thymidylate synthase (TS 2R3R) and reduced folate carrier (RFC 80G>A) were detected by PCR-RFLP. Results. 57% of the patients had prolonged increased serum levels of MTX in at least 1 course. 59% patients had the MTX courses at the scheduled time, 35% had one course postponed and 6% 2 courses. Mucositis was observed in 22% patients after 1 out of 3 courses, 19% after 2 and 20% after 3 courses. 28% patients had 1 transfusion, 6% had 2 and 1% had 3 transfusions during the MTX courses. Transaminases were elevated 2 times above the normal upper level in 25% of the patients. Skin rash was observed in 32% patients. Using Mann Whitney analysis we found a significant relation between polymorphisms of the MTHFR 1298A>C and for the MTRR 66A>G genes and side effects. The MTHFR 1298A>C polymorphism was related to less transfusions (p<0.05). Recovery of WBC count in HR ALL patients tended to be slower in patients with a MTHFR 1298AA genotype (P=0.053). HR patients with a MTRR 66AG genotype had a slower recovery of platelet count (p=0.004) Conclusion. Common polymorphisms in the MTHFR (677C>T), MS, SHMT, TS, RFC, and MTHFD1 genes are not related to the occurrence of side effects of MTX treatment in childhood ALL. Polymorphisms in the MTHFR (1298A>C) and MTRR (66A>G) gene are related to hematological toxicity.