Background & Aims: Tenofovir disoproxil fumarate (TDF) is one the most potent nucleot(s)ide analogues for treating chronic hepatitis B virus (HBV) infection. Phenotypic resistance caused by genotypic resistance to TDF has not been reported. This study aimed to characterize HBV mutations that confer tenofovir resistance. Methods: Two patients with viral breakthrough during treatment with TDF-containing regimens were prospectively enrolled. The gene encoding HBV reverse transcriptase was sequenced. Eleven HBV clones harboring a series of mutations in the reverse transcriptase gene were constructed by site-directed mutagenesis. Drug susceptibility of each clone was determined by Southern blot analysis and real-time PCR. The relative frequency of mutants was evaluated by ultra-deep sequencing and clonal analysis. Results: Five mutations (rtS106C [C], rtH126Y [Y], rtD134E [E], rtM204I/V, and rtL269I [I]) were commonly found in viral isolates from 2 patients. The novel mutations C, Y, and E were associated with drug resistance. In assays for drug susceptibility, the IC50 value for wild-type HBV was 3.8 +/- 0.6 mu M, whereas the IC50 values for CYE and CYEI mutants were 14.1 +/- 1.8 and 58.1 +/- 0.9 mu M, respectively. The IC90 value for wild-type HBV was 30 +/- 0.5 mu M, whereas the IC90 values for CYE and CYEI mutants were 185 +/- 0.5 and 790 +/- 0.2 mu M, respectively. Both tenofovir-resistant mutants and wild-type HBV had similar susceptibility to the capsid assembly modulator NVR 3-778 (IC50 < 0.4 mu M vs. IC50 = 0.4 mu M, respectively). Conclusions: Our study reveals that the quadruple (CYEI) mutation increases the amount of tenofovir required to inhibit HBV by 15.3-fold in IC50 and 26.3-fold in IC90. These results demonstrate that tenofovir-resistant HBV mutants can emerge, although the genetic barrier is high. Lay summary: Tenofovir is the most potent nucleotide analogue for the treatment of chronic hepatitis B virus infection and there has been no hepatitis B virus mutation that confers > 10-fold resistance to tenofovir up to 8 years. Herein, we identified, for the first time, a quadruple mutation that conferred 15.3-fold (IC50) and 26.3-fold (IC90) resistance to tenofovir in 2 patients who experienced viral breakthrough during tenofovir treatment. (C) 2019 Published by Elsevier B.V. on behalf of European Association for the Study of the Liver.