Adefovir dipivoxil has been used alone or together with lamivudine to suppress lamivudine-resistant hepatitis B virus (HBV). However, the dynamics of HBV populations under different selection pressures and their impact on treatment outcome are poorly understood. Pyrosequencing® was applied to quantify longitudinally the evolution of wild type and lamivudine/ adefovir-resistant HBV. Eight patients, with lamivudine-resistant HBV, were randomized to receive adefovir monotherapy or adefovir/lamivudine combination therapy for a median of 79 and 71 weeks, respectively. Pyrosequencing® proved highly sensitive with a lower limit of quantitation of minor HBV populations of 2% irrespective of viraemia levels. Adefovir/lamivudine treatment resulted in greater viraemia reduction than adefovir monotherapy. During combination therapy, lamivudine-resistant HBV populations (codons 180 and 204) remained dominant (>90%) and no adefovir-resistance developed. During adefovir monotherapy, reversion to wild-type HBV was detected in two patients with one patient accumulating rapidly adefovir-resistant HBV along with increased viraemia. In conclusion, the dynamics of drug-resistant HBV strains vary under different selection pressures which have a significant impact on the success of rescue therapy, as well as for the selection of new mutations. The use of techniques such as Pyrosequencing provides an evidence-based approach for successful management of drug-resistant HBV. J. Med. Virol. 80: 1160–1170, 2008. © 2008 Wiley-Liss, Inc.