The aim was to study the seroprevalence of Hepatitis C virus (HCV) infection and related risk factors in Aseer region in southwestern Saudi Arabia, the region known to be of the highest endemicity of viral hepatitis. In a cross-sectional study, all participants were interviewed using structured questionnaire. HCV infection was diagnosed using fourth-generation ELISA. All positive and equivocal HCV serology results were further confirmed by using a qualitative confirmatory RT-PCR. The study enrolled 10,234 participants. A seroprevalence of 2.2% (95% CI: 1.9-2.5%) was found. In multivariate logistic regression analysis, the study showed that males had significantly more risk to become seropositive for HCV (aOR = 1.437, 95% CI: 1.071-1.927) compared to females. Similarly, participants having history of blood transfusion had more than two times the risk of becoming seropositive for HCV (aOR = 2.079, 95% CI: 1.037-4.149). HCV infection in the study area is still high in the plateau phase. It is recommended to have an active educational and media campaign about the risks of HCV infections. Workshops and training of qualified laboratory staff related to blood banking seem mandatory. (C) 2016 Wiley Periodicals, Inc.