Iron overload is a significant cause of morbidity and mortality for patients who require frequent transfusions. We completed a prospective, cross-sectional study to evaluate the prevalence of iron overload in previously transfused childhood cancer survivors. Survivors recruited from the University of Minnesota Long-Term Follow-Up Clinic were stratified into 3 groups: oncology patients not treated with hematopoietic stem cell transplantation (HSCT) (n=27), patients treated with allogeneic HSCT (n=27), and patients treated with autologous HSCT (n=9). Serum ferritin was collected and hepatic magnetic resonance imaging (FerriScan) was obtained for those with iron overload (defined as ferritin ≥1000 ng/mL). The prevalence of iron overload in subjects with a history of allogeneic HSCT was 25.9% (95% CI, 9.4%-42.5%) compared with only 3.7% (95% CI, 0%-10.8%) in subjects treated without HSCT and 0% in subjects treated with autologous HSCT. No association was found between serum ferritin levels and the presence of cardiac, liver, or endocrine dysfunction. The prevalence of iron overload in subjects who received no HSCT or autologous HSCT is low in our study. A higher prevalence was found in patients receiving allogeneic HSCT, reiterating the importance of screening these patients for iron overload in accordance with the current Children’s Oncology Group Long Term Follow-Up Guidelines.