Hepatitis B virus (HBV) is an enveloped virus with a small (3.2-kb) partially double-stranded DNA genome that causes acute and chronic infections. The impact of these infections on public health worldwide is enormous, with an estimated prevalence of 2 billion acute infections and 360 million chronic infections globally. This review focuses on chronic hepatitis B and the molecular assays used in its diagnosis and management. Background information, including that about features of the hepatitis B virion, viral replication, and epidemiology of infection, that is important for understanding chronic hepatitis B and molecular diagnostic tests for HBV is provided. To facilitate an understanding of the utility of molecular testing for chronic hepatitis B, the four stages of chronic hepatitis B infection that are currently recognized, as well as an additional entity, occult hepatitis B, that can be diagnosed only by sensitive nucleic acid amplification methods, are reviewed in detail, including available therapeutic agents. The molecular diagnostic content focuses on tests for HBV DNA quantification, genotyping, and mutation detection (including precore/core promoter and antiviral resistance mutations). The discussion of these tests encompasses their current utility and performance characteristics, drawing from current clinical guidelines and other studies from the literature. In recognition of the continual evolution of this field, the final section describes emerging molecular markers with future diagnostic potential.