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Occult hepatitis B virus (HBV) is defined as low-level HBV DNA without hepatitis B surface antigen (HBsAg). Prevalence estimates vary widely. We determined the prevalence of occult HBV at the University of Cincinnati Infectious Diseases Center (IDC).Patients in the IDC HIV database (n = 3867) were randomly selected using a 25% sampling fraction. Samples were pooled for HBV nucleic acid extraction. Pools were tested for HBV DNA by a real-time polymerase chain reaction (PCR) assay to coamplify core/surface protein regions. The PCR assay was run on all individual samples from each DNA+ pool. DNA+ samples were tested for HBV serologic markers.A total of 909 patients without known HBV were selected. The mean CD4 count was 384 cells/mm3. Forty-three patients were HBV DNA+. Twelve of 43 were DNA+/HBsAg− (95% confidence interval for database: 0.58% to 1.90%). Five of 12 were negative for all serologic markers. Alanine aminotransferase, aspartate aminotransferase, and HBV DNA titers were elevated in HBsAg+ patients versus occult patients and versus HIV-monoinfected patients. No other significant differences were detected. No occult HBV patient was on treatment with anti-HBV activity.Forty-three percent of those with HBV were not previously identified as HBV+, indicating the need for ongoing screening in high-risk populations. Occult HBV may occur in persons with all negative serologic markers, representing a challenge for identification.