Ultra-deep pyrosequencing (UDPS) was used to analyse the dynamics of quasispecies and resistant mutations during telbivudine (LDT) treatment of hepatitis B patients. Twenty-six HBeAg-positive chronic hepatitis B patients were treated with LDT for a period of 104 weeks and were characterized as 16 responders, six partial responders and four viral breakthrough patients based on hepatitis B virus (HBV) DNA levels. The plasma samples were subjected to UDPS of the reverse transcriptase (RI) region of HBV. Mutations rtM2041, rtL80I and rtL80V were detected in at least three of the four viral breakthrough patients, indicating the significant roles of the mutations in resistance to LDT. The degree of complexity of viral quasispecies remained in a steady state in the absence of selection pressure, but increased after the LDT treatment. The complexity in the responder group at week 12 was significantly higher than that in the group comprising partial responders and viral breakthrough patients. In vitro replication efficiency analyses showed that the RI mutations had different impacts on HBV replication, with a tendency of rtM2041>rtL80V>rtL801. Furthermore, double mutations rtL80I/M204I and rtL80V/M204V had replication efficiency similar to that of rtL80I and rtL80V, respectively. Consistent with previous studies, mutation rtM2041 was found to be highly resistant to LDT. However, in contrast with their sensitivity to lamivudine, rtL80I and rtL8OV were moderately resistant to LDT. Our results indicated that rtL80I and rtL8OV may not only serve as replication complementary mutations to rtM2041, but also directly contribute to the LDT resistance.