The emergence of resistance mutations in the reverse transcriptase gene of hepatitis B virus (HBV) is associated with treatment failure. Entecavir (ETV) is one of the most potent anti-HBV reagents; it has a very low resistance rate and is used as the first-line treatment for chronic hepatitis B. In this study, we isolated HBVs in 4 ETV-refractory patients (2 with viral breakthrough, 1 with partial virological response, and 1 with flare-up) and assessed ETV resistance using replication-competent 1.38-fold HBV genome-length molecular clones. The full genome sequences of infected HBVs in ETV-refractory patients were determined. The HBV molecular clones were generated with the patient-derived sequences. After transfection of these molecular clones into HepG2 cells, viral replications and ETV susceptibilities were evaluated by measuring the amount of intracellular core-particle-associated HBV DNA using Southern blotting and real-time polymerase chain reaction. Among these cases, ETV-resistant variants were detected in 2 patients with viral breakthrough and responsible amino acid mutations in reverse transcriptase were successfully identified in these variants. No ETV-resistant mutation was detected in the other cases. The identified ETV-resistant mutations did not confer resistance to tenofovir disoproxil fumarate. Conclusion: The HBV replication model with patient-derived sequences is useful for assessing replication efficiency, susceptibility to anti-HBV reagents, and responsible resistance mutations and can aid in choosing the appropriate treatment strategy for treatment-failure cases of chronic hepatitis B. (Hepatology Communications 2017;1:110-121).