Entecavir (ETV) is a potent nucleoside analogue against hepatitis B virus (HBV). Because of development of ETV resistance requires at least three amino acid substitutions in HBV polymerase (pol) gene, emergence of ETV resistance is rare (similar to 1%) in nucleoside-naive patients after up to 5 years of treatment. However, it has been suggested that lamivudine (LAM) therapy can preselect for HBV variants associated with resistance to ETV treatment. ETV resistance increased to 51% of patients after 5 years of ETV treatment in LAM refractory patients. The diagnosis of ETV resistance in chronic hepatitis B patients, mainly based on four types of molecular assays: direct sequencing, line probe assay, clonal analysis, and restriction fragment length polymorphism (RFLP) analysis. The applications of other assays are currently more specialized, and their use is more limited. The utility of these assays and their performance characteristics are reviewed below. Briefly, the monitoring of drug-resistant variants is important in the elucidation of the prevalence and mechanisms of resistance development and for the more effective management of treatment options.