Background and Objective: Presence of hepatitis B core antibody (anti-HBc) in the absence of hepatitis B surface antigen (HBsAg) and hepatitis B surface antibody (anti-HBs) is defined, as isolated anti-HBc. little is known about the clinical significance of the isolated anti-HBc in hepatitis B virus (HBV) infections. The aim of this study was to assess the significance of anti-HBc as the only marker of HBV infection in high risk patients. Patients and Methods: In this cross-sectional study, 395 patients including 289 patients on chronic hemodialysis (HD) and 106 HIV infected subjects were enrolled. HBsAg, anti-HBs, anti-HBc, Hepatitis C antibody (anti-HCV) and Alanine aminotransferase (ALT) levels were tested in all subjects. The presence of HBV-DNA was determined quantitatively in plasma samples of patients with isolated anti-HBc by real-time PCR. Results: Of 395 patients, 40 (10.13%, 95% CI, 7.1%-13.1%) had isolated anti-HBc. HBV-DNA was detectable in 12 of 40 patients (30%, 95% CI, 15.8%-44.2%) who had isolated anti-HBc. Conclusion: Our study showed that detection of isolated anti-HBc could reflect unrecognized HBV infection; hence, screening of these patients is useful to preventing of HBV transmission.