Seven hundred nucleoside treatment-naive patients were enrolled in two phase 3 trials of adefovir dipivoxil (ADV) for the treatment of chronic hepatitis B. To monitor for the emergence of potential adefovir resistance mutations over the first 48 weeks, all intent-to-treat patients (467 ADV-treated and 228 placebo patients) were included in a prospectively defined, treatment-blinded, virology substudy. The study protocol mandated genotypic analysis for all patients with detectable hepatitis B virus (HBV) DNA by Roche Amplicor polymerase chain reaction (PCR) at baseline and week 48, and in vitro phenotypic analyses for patients with conserved site substitutions in HBV polymerase or 1.0 log10 or greater increase in HBV DNA from nadir. Paired sequences of the entire HBV reverse transcriptase were obtained for 271 ADV-treated and 227 placebo patients by using a sequencing method that detects down to 30% of minor species present within mixtures. Four substitutions (rtS119A, rtH133L, rtV214A, and rtH234Q) developed once each at conserved sites in HBV polymerase in 4 ADV-treated patients. Seven conserved site substitutions developed in 6 placebo patients. HBV mutants encoding the 4 substitutions that emerged in ADV-treated patients remained fully susceptible to adefovir in vitro. Furthermore, these 4 ADV-treated patients had HBV-DNA reductions of 3.3 to 5.9 log10 copies/mL by week 48 with no rebound. All other substitutions occurred at very low frequencies (<1.6%) at polymorphic sites and were not associated with HBV-DNA increases in patients or adefovir resistance in vitro. In conclusion, no adefovir resistance mutations were identified in a large group of chronic hepatitis B patients treated with ADV for 48 weeks.