Serological detection of isolated anti-hepatitis B core antibody (anti-HBc) can occur in various scenarios, but the most clinically relevant situation is occult hepatitis B virus (HBV) infection (OBI). The purpose of this study was to evaluate the prevalence and clinical relevance of isolated anti-HBc and of OBI with isolated anti-HBc from an unselected hospital population. A total of 14,253 patients referred for hepatitis B surface antigen (HBsAg)/anti-HBs testing were classified into either the Health Promotion Center (HPC) group or the patient group. For patients who were negative for both HBsAg and anti-HBs, an anti-HBc test was additionally performed. An HBV DNA real-time PCR test was performed on isolated anti-HBc patients, and their demographic and clinical data were reviewed. The measured prevalence of isolated anti-HBc and OBI in isolated anti-HBc patients was 5.9 % and 4.7 %, respectively. Prevalence was higher in males, elderly people, and the patient group than in females, the younger, and the HPC group, respectively. In most cases, the levels of HBV DNA load were very low (less than 200 IU/mL), and most cases of OBI presented without liver disease or history of HBV infection. The prevalence of isolated anti-HBc and OBI are affected by the methods of detection, subject population, and constituent factors such as sex and age. Although detection of HBV DNA does not always indicate infectivity, highly sensitive standardized HBV DNA tests are needed in clinical settings to exclude the possibility of OBI, especially in the advanced age group.