Considering the potential risks associated with occult hepatitis B virus (HBV) infection, this study was designed to investigate the magnitude and genotypic pattern of occult HBV infection among hemodialysis patients. All patients on regular hemodialysis attending the dialysis centers located in southern Iran and 277 nonhemodialysis controls were invited to participate in this study. Serum samples were tested for detection of hepatitis B core antibody (HBcAb) and hepatitis B surface antigen (HBsAg) by competitive enzyme immunoassay and sandwich ELISA, respectively. The molecular evaluation of HBV infection was conducted by two nested polymerase chain reaction (PCR) assays, targeting S, X, and precore regions of HBV genome, and sequencing by Sanger dideoxy sequencing technology. Moreover, HBV viremic samples were tested for hepatitis C virus (HCV) coinfection by HCV Ab ELISA and seminested reverse transcriptase PCR. Of 279 hemodialysis patients, five (1.8%) were positive for HBsAg, 66 (23.7%) were positive for HBcAb, and 32 (11.5%) had HBV viremia with HBV genotype D, sub-genotype D3 and subtype ayw2. Moreover, 90.6% of the hemodialysis patients with HBV viremia had occult HBV infection. Hemodialysis patients (11.5%) had significantly higher prevalence of HBV viremia than nonhemodialysis controls (1.08%; P = 0.0001). The prevalence of HBV viremia among hemodialysis patients was not statistically associated with duration of hemodialysis, age and gender distribution. In contrast, HBV viremia was significantly associated with place of residency and ethnicity, with residents of Dashtestan and Arab having had significantly higher prevalence of HBV viremia compared with the residents of other cities and Fars patients. Notably, 27.6% and 6.9% of hemodialysis patients infected with occult HBV infection were also positive for anti-HCV antibodies and HCV viremia, respectively. High prevalence of occult HBV infection was detected in hemodialysis patients, whereas 62% of patients with occult HBV infection were negative for HBcAb. Therefore, screening of all hemodialysis patients by sensitive molecular tests, regardless of the pattern of HBV serological markers, is recommended to increase the diagnosis rate of HBV infection.