Objectives: To investigate the prevalence, genotype distribution, and risk factors of hepatitis C virus (HCV) infection among patients undergoing regular hemodialysis in Bushehr province in southern Iran. Methods: All chronic hemodialysis patients from the cities of Dashtestan, Genaveh, and Bushehr participated in this study. Enzyme-linked immunosorbent assay was used to detect anti-HCV antibodies. Molecular detection of HCV infection was performed by semi-nested reverse transcription polymerase chain reaction assay, targeting 5' untranslated region and core region of the genome, and sequencing. Results: Of 279 hemodialysis patients, 15 (5.4%) were positive for anti-HCV antibodies, and two (0.7%) patients had HCV viremia with genotype 3a. The hemodialysis patients had a significantly higher seroprevalence of HCV than the control group (p = 0.007). Patients with Arab ethnicity had significantly higher anti HCV seroprevalence compared to those with Fars ethnicity (p = 0.026). Anti-HCV seropositivity was not statistically associated with the patients' sex, age group, place of residence, level of education, duration of hemodialysis, or history of blood transfusion. Conclusions: Considering the high seroprevalence of HCV in hemodialysis patients, regular screening of these patients for HCV infection and prompt treatment of those found infected are recommended.