Current therapies for chronic hepatitis B virus (HBV) infection do not provide adequate long-term control of viral replication in the majority of patients. Monotherapy with nucleoside analogs, such as lamivudine and famciclovir, is effective for short periods but results in the emergence of drug-resistant HBV in a substantial number of patients within 1 year of therapy. Adefovir dipivoxil (ADV) has demonstrated clinical activity against wild-type and lamivudine-resistant HBV, but it is unclear whether resistance mutations will emerge after long-term therapy with this drug. To determine whether extended treatment with ADV led to the emergence of drug-resistant populations of HBV, we analyzed virus isolated from patients currently enrolled in a long-term open-label study. The reverse transcriptase domain of HBV polymerase was amplified and sequenced from patients that had received a cumulative exposure of up to 60 weeks of ADV. During our analyses, several previously unreported amino acid substitutions were observed in the reverse transcriptase domain of HBV. Importantly, none of the observed mutations occurred in more than 1 patient, nor were they associated with an adefovir-resistant phenotype in vitro. Furthermore, none of the patients from whom these mutant viruses were isolated had evidence of virologic rebound. In conclusion, these results, although based on a limited number of patients, suggest that treatment with ADV does not lead to the emergence of resistant virus after up to 60 weeks of therapy.