Hepatitis C virus infection is a major health problem worldwide. The current study estimated seroprevalence of Hepatitis C virus (HCV) and evaluated associated factors among volunteer blood donors of the Northwest Frontier Province (NWFP), Pakistan. Of 1,131 volunteer blood donors enrolled, 46 (4.1%) were positive for anti-HCV antibodies. Multivariate logistic regression analysis revealed that positive donors were more likely to be 27-32 years old or >32 years old, have had 1-2 injections or >2 injections in the past year, or 1-5 intravenous (IV) drips or >5 I/V drips in the past 5 years. Positive donors had a family history of jaundice and were more likely to have been shaved (facial and armpit) by barbers. There was high prevalence of anti-HCV antibodies among blood donors of the NWFP. Public awareness programs should target the identified risk factors to prevent HCV transmission. We highlight the weakness of the health care system for blood donation, as it does not offer any record management for donors.