Background: β-thalassemia trait is one of the most common genetic disorders in Mediterranean countries. Previous studies have shown that β- thalassemia trait has a protective effect against malaria, coronary artery disease, hypertension and Alzheimer disease. We hypothesize that due to the shorter life span of red blood cells and increased hematopoiesis, these patients are at increased risk of developing hematological malignancies. Thus, this study investigated the possible effect of β-thalassemia trait on childhood malignancies.Methods: This was a case-control study that included 432 children with malignancies (leukemias and lymphomas as well as solid tumors) as the case group and 432 healthy individuals who referred for premarital β-thalassemia trait screening as the control group. All patients underwent a complete blood count as well as hemoglobin electrophoresis. Hemoglobin A2≥3.5% or hemoglobin F between 2% and 10% were considered diagnostic for β- thalassemia trait. The results were compared between groups with the chi-squared test.Results: Mean age of the patients was 7.08 ± 5.1 years and mean age in the control group was 25.46 ± 7.3 years. There were 253 (58.6%) boys and 179 (41.4%) girls among the patients and 308 (71.3%) men and 124 (28.7%) women among the controls. The prevalence of β-thalassemia trait was 5.6% in the case group and 11.3% in the control group (P=0.001). The mean hemoglobin level was 9.81 ± 3.1 g/dL in those with malignancies and 15.3 ± 1.7 g/dL in healthy individuals (P<0.001).Conclusion: β-thalassemia trait is a protective factor against developing childhood malignancies in an unselected Iranian population. However, more studies are needed to clarify this issue.