Background: Regular blood transfusion and iron chelation therapy have improved the quality of life of patients with thalassemia and increased their longevity, but transfusion also increases the frequency of endocrine complications, possibly because of iron deposition in the pituitary gland or the gonads, or both. Objective: To evaluate testicular function in patients with thalassemia major by basal hormonal study, and identify risk factors for dysfunction. Methods: We performed a cross-sectional study of 28 patients with thalassemia major aged 11.7 ± 1.8 (8−14.9) years (15 in prepuberty, 13 in puberty with no delayed puberty) who had regular blood transfusions. A normal control group comprised 64 boys who were matched for age and Tanner genital stage. Results: The mean level of serum ferritin in the previous year was 1,575 ± 642 ng/mL, and the onset of blood transfusion was at 3.8 ± 2.3 years and iron chelation therapy was 6.6 ± 2.8 years. The trend for anti-Müllerian hormone levels in patients and controls was similar with age, and although higher in the patients, particularly at Tanner stage II, was not significantly different. Testosterone levels were lower in the patients compared with controls; particularly at Tanner stages IV-V (290.88 vs. 537.4 ng/dL, P < 0.05). Serum follicle-stimulating hormone and luteinizing hormone levels were not significantly different between the groups at any Tanner stage. Conclusion: Patients who received regular blood transfusions had normal Sertoli cell function. Leydig cell dysfunction may occur, even though the patients had a normal pubertal onset.