Hepatitis B (HBV) and C (HCV) virus infection are the most important infections transmitted by the parenteral route in hemodialysis patients. This study is the first report of prevalence of viral hepatitis and hepatitis C virus genotypes in southwest Iran among hemodialysis patients. A cross-sectional study was carried out among 214 hemodialysis patients of the Central hemodialysis unit, from March 2005 to August 2006. Serum samples were tested for HBsAg and anti-HCV using specific enzyme linked immunoassay (ELISA) kits and confirmed by PCR (HBV) and RT PCR (HCV). HCV genotypes were determined with HCV genotype specific primers using HCV genotype kit. Out of 214 hemodialysis patients, 34 were positive for anti-HCV (7.9%, 95% CI: 4.32-11.56) and 11 for HBsAg (5.1%, 95% CI: 2.18-8.1). The duration of treatment by hemo-dialysis was significantly associated with HBV and HCV positivity (P< 0.001). The predominant HCV genotype in the region was 1a (41.1%, 7/17), whilst genotypes 3a and 1b were found in 35.2% (6/17) and 23.5% (4/17) subjects, respectively. In conclusion although anti-HCV and HBsAg positivity in hemodialysis patients in Khuzestan province are smaller than those found in some other Iranian provinces and neighboring countries, they are still high. Enforcement of universal precautions in infection control, routine testing of patients, and serial determination of hepatic enzymes should be the common practice in dialysis centers in Iran.