Aims: Tenofovir disoproxil fumarate (TDF) is the most potent nucleoside analog for the treatment of chronic hepatitis B virus (HBV) infection. Genotypic resistance to tenofovir has not yet been reported. This study aimed to characterise HBV mutations that confer tenofovir resistance. Methods: Two consecutive patients with viral breakthrough during treatment with TDF-containing regimens were prospectively enrolled. The gene encoding HBV reverse transcriptase (RT) was sequenced. Nine HBV clones harbouring a series of mutations in the RT gene were constructed by site-directed mutagenesis. Drug susceptibility of each clone was determined by Southern blot analysis and real-time PCR. Relative frequency of mutants were evaluated by ultra-deep sequencing. Results: (Please understand that actual mutation site numbers were replaced by bold, underlined alphabetical letters since they are now confidential due to embargo policy.) Seven mutations (rtSaaaC [C], rtHbbbY [Y], rtDcccE [E], rtVdddL, rtLeeeM, rtMfffV, and rtLgggI) were commonly found in viral isolates from both patients after viral breakthrough; C, Y, and E were novel mutations. An HBV mutant harbouring all three mutations (CYE) was resistant to tenofovir. The IC50 values for wild-type HBV and the CYE mutant were 3·8 ± 0·6 μM and 14·1 ± 1·8 μM, respectively. Ultra-deep sequencing showed that CYE mutant was dominant than any other mutant in both patients. All tenofovir-resistant mutants had similar susceptibility to a core inhibitor, NVR 3-778 (IC50 < 0·4 μM) compared with wild-type (IC50 = 0·4). Conclusions: Our study reveals that a novel triple mutation (CYE) is associated with tenofovir-resistance. These results demonstrate that tenofovir-resistant HBV mutants can emerge, although the genetic barrier is high. A novel core inhibitor might be a potential rescue therapy for tenofovir-resistant HBV.