Blood-borne viruses including Hepatitis B and C, HIV, HTLV-1 and parvovirus B19 are still a factor of concern, especially for hemophilia patients. Although the safety of the blood supply continues to improve worldwide, the blood supply system in Afghanistan was damaged by many years of conflict and political instability. To date, there are few studies focused on the prevalence of blood-borne viruses in hemophilia patients. This study is first to investigate the prevalence of five blood-borne viruses in Afghanistan hemophilia patients in four cities including Kabul, Herat, Mazar-i-Sharif and Jalal Abad. A total of 80 hemophilia male patients were screening for the presence of five transfusion-transmitted viruses using ELISA and PCR. Data obtained showed 2.5% seropositivity for HBV, 8.75% seropositivity for HCV, and 91.25% seropositivity for parvovirus B19. None of the patients were positive for HIV and HTLV-1 and the prevalence of HCV was higher in older patients rather than younger patients. This finding, the first to report in Afghanistan, shows a high prevalence of parvovirus B19 in Afghanistan hemophilia patients and implementation of highly sensitive screening is necessary.