Background/Aims: Data on the prevalence of hepatitis C virus (HCV) infection in Libya, and particularly among hemodialysis patients, are scarce. The aim of this study was to assess the prevalence of HCV infection among hemodialysis patients at 37 dialysis centers in Libya and review the demographic features in this group of patients. Patients and Methods: The present cross-sectional study included a total of 2325 patients who had spent at least 3 months in hemodialysis at 37 dialysis centers in different cities across the three main regions of Libya (Western, Eastern, and Southern regions). There were 1028 (44.2%) female and 1297 (55.8%) male with mean age 53.4 ± 15 years. Hypertension (36.4%), diabetes mellitus (33.3%), renal diseases (10.9%), genetic diseases (7.5%), and unclear reasons (12%) were diverse underlying causes of end-stage renal disease in these patients. Anti-HCV antibody was screened by a third-generation enzyme-linked immunosorbent assay technique. Results: The overall seroprevalence of HCV infection among hemodialysis patients in Libya was 16.7% (388/2325), ranging from 0% to 26.3% at different centers. The overall frequency of anti-HCV antibodies was higher in Western region (18.9%) than Eastern (18.3%) and Southern (6.1%) regions of Libya. It is noteworthy that the prevalence of HCV was 0% in numbers of dialysis centers across the three regions. The duration of hemodialysis was significantly longer in HCV-positive hemodialysis patients than in HCV-negative patients (P = 0.01). Hepatitis B surface antigen was positive in 92 hemodialysis patients (4%), of them 8 patients (8.7%) had coinfection with HCV infection. Conclusion: The present study showed an intermediate-prevalence rate of HCV infection among hemodialysis patients in Libya. The relatively low prevalence of HCV infection in the general Libyan population and HCV infection associated with a longer duration of hemodialysis indicate nosocomial transmission due to inappropriate infection control practices as the main HCV route of infection in these health-care settings.