Background and Aims: Hepatitis C is a blood-borne virus that causes a liver inflammation. The prevalence of hepatitis C (HCV) in hemodialysis patients is more than the general population. Periodic screening of HCV in dialysis centers is necessary to control this viral disease. The aim of this study is the evaluation of HCV infection among hemodialysis patients living in Yazd province of Iran. Materials and Methods:In this cross sectional study, two milliliters blood sample has been taken from each of 188 hemodialysis patients. Third-generation enzyme-linked immunosorbent assay (ELISA) and RT-PCR methods were carried out for detecting HCV antibody and viral genome in patient sera. Demographic and clinical characteristics of patients were collected and data were analyzed by Fisher's exact test and Mann-Whitney U test in SPSS software v.19. Results: HCV-Ab was positive in 12 hemodialysis patients. Eight cases had a history of blood transfusion and four patients had a history of kidney transplantation. Anyone didn't have a history of skin tattooing or intravenous drug usage. HCV-RNA was positive in 4 cases and was negative in 8 patients with positive HCV-Ab. Conclusion: HCV prevalence using ELISA and RT-PCR tests among hemodialysis patients living in Yazd provenance of Iran is 6.3% and 2.1%, respectively. Risk factors are the hemodialysis duration (p<0.001) and kidney transplantation (p=0.005).