BACKGROUND: Though regular blood transfusion improves the overall survival of patients with beta-thalassemia, it carries a definite risk of infection with blood-borne viruses. We carried out this multicenter study to provide epidemiologic data on hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) infection among Iranian beta-thalassemic patients. Moreover, HCV infection-associated risk factors were investigated in this population. METHODS: Seven hundred and thirty-two patients with beta-thalassemia major or beta-thalassemia intermedia, selected from five provinces of Iran including Tehran (n = 410), Kerman (n = 100), Qazvin (n = 95), Semnan (n = 81), and Zanjan (n = 46), were enrolled in this study. Using ELISA, their sera were tested for HBsAg, HBcAb, HBsAb, HCVAb, and HIVAb. The positive HCVAb results were confirmed by RIBA-2nd generation. RESULTS: The study sample consisted of 413 males and 319 females, with a mean +/- SD age of 17.9 +/- 9.0 years. One hundred forty-one (19.3%) patients were HCVAb positive; 11 (1.5%) were HBsAg positive. No one was HIVAb positive. Univariate analysis showed that beta-thalassemia major (P = 0.01), older age (P = 0.001), longer transfusion duration (P = 0.000), HBsAg seropositivity (P = 0.03), and higher serum ferritin level (P = 0.002) were significantly associated with a higher prevalence of HCV. Furthermore, the prevalence of HCV infection dropped significantly after the implementation of blood donors screening (22.8% vs. 2.6%; P = 0.000). Using multivariate analysis, beta-thalassemia major (P = 0.002), age (P < 0.001), serum ferritin level (P < 0.001), as well as consumption of unscreened blood (P = 0.003), were independent factors associated with HCV infection. CONCLUSION: The prevalence of HCV infection is much higher among Iranian beta-thalassemic patients as compared with HBV and HIV infections. Routine screening of donated blood for HCV is highly recommended.