Transfusion-transmitted viral infections, including hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiency virus (HIV) infections continue to be a major challenge in hemophilia patient. This study was conducted to assess the seroprevalence of HBV, HCV, and HIV infections among Iranian hemophilia patients. A cross-sectional study was conducted on 580 hemophilia patients (mean age ± SD = 27.11 ± 14.9) referred to the Namazi hospital, Shiraz University of medical sciences, Shiraz, Iran during July 2012 to August 2015. Blood samples were taken from the patients and tested for HBV surface antigen (HBsAg), antibody to HBV surface antigen (anti-HBs), antibody to HBV core antigen (anti-HBc), anti-HCV antibody (HCVAb), and anti-HIV antibody (HIVAb). The association of HBV, HCV, and HIV infections with age, sex, type of hemophilia (A or B), severity of hemophilia, blood group system, history of blood transfusion, and surgery were statistically analyzed using SPSS software and chi-square/Fisher exact tests. The seroprevalence of HCVAb and HBsAg were 11.4 % (66/ 580) and 0.2 % (1/580), respectively. None of the hemophilia patients were positive for HIV antibody. There was a significant association between HCV seropositivity and age (P < 0.001), severity of hemophilia (P < 0.0001), and history of blood transfusion (P = 0.050). It seems that strategies for prevention of HBV and HIV infections have been successful in south of Iran, but HCV is still a major infection of concern in multi-transfused patients.