Reverse transcriptase (RT) mutations contribute to hepatitis B virus resistance during antiviral therapy with nucleos(t)ide analogs. However, the composition of the RT quasispecies and their interactions during antiviral treatment have not yet been thoroughly defined. In this report, 10 patients from each of 3 different virological response groups, i.e., complete virological response, partial virological response and virological breakthrough, were selected from a multicenter trial of Telbivudine treatment. Variations in the drug resistance-related critical RT regions in 107 serial serum samples from the 30 patients were examined by ultra-deep sequencing. A total of 496,577 sequence reads were obtained, with an average sequencing coverage of 4,641X per sample. The phylogenies of the quasispecies revealed the independent origins of two critical quasispecies, i.e., the rtA181T and rtM204I mutants. Data analyses and theoretical modeling showed a cooperative-competitive interplay among the quasispecies. In particular, rtM204I mutants compete against other quasispecies, which eventually leads to virological breakthrough. However, in the absence of rtM204I mutants, synergistic growth of the drug-resistant rtA181T mutants with the wild-type quasispecies could drive the composition of the viral population into a state of partial virological response. Furthermore, we demonstrated that the frequency of drug-resistant mutations in the early phase of treatment is important for predicting the virological response to antiviral therapy.