The development of serological assays to detect hepatitis B surface antigen (HBsAg) has played a major role in the diagnosis of hepatitis B virus (HBV) infection. With other hepatitis B serological assays, a diagnosis of acute or chronic HBV infection, past infection, or successful vaccination can be determined. However, serological profiles can at times be atypical or ambiguous. Some of these difficulties may be overcome by HBV DNA testing, which may elucidate an individual's true hepatitis B status. In addition, quantification of HBV DNA provides a means of monitoring the effectiveness of antiviral therapy and detecting the early development of antiviral drug resistance. Molecular diagnostics are also being applied to HBV-infected liver tissue. The use of molecular techniques to quantify intrahepatic HBV DNA and other key HBV replicative intermediates may provide additional options for monitoring and predicting treatment efficacy, but such assays require further validation before they can be used routinely.