Hepatitis B virus causes horizontally transmitted infectious hepatopathy of primates and may progress to hepatocellular carcinoma. Historically, a small number of chimpanzees (Pan troglodytes) living in accredited North American zoos have been confirmed with positive hepatitis B serology consistent with exposure. However, the overall status for this population and the interpretation of these individual test results have not been established previously. The current U.S. zoo-housed population (n = 259) was assessed serologically for hepatitis B by surface protein antigen (HbsAg) and surface antibodies (anti-Hbs). Signalment, origin, current health status, history of liver disease, and hepatitis B vaccination history were obtained for each animal. Serologic status was measured directly in 86.5% (n = 224) of these individuals, with 2.2% (n = 5) of the study population determined to be chronically infected by positive HbsAg and negative anti-Hbs status. Additionally, 11.6% (n = 26) of the directly measured population tested were HbsAg negative and anti-Hbs positive, which was indicative of viral exposure. No animals were determined to be acutely infected as HbsAg and anti-Hbs positive. Although these results demonstrated a relatively low prevalence of hepatitis B infection among these chimpanzees, the varied serologic results between institutions underscored the importance of routine serologic testing, especially at times of proposed transfers, and consideration of species vaccination protocols.