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.