BACKGROUND & AIMS: Patients chronically infected with the hepatitis B virus (HBV) and receiving long-term treatment with nucleoside or nucleotide analogues are at risk of selecting HBV strains with complex mutational patterns. We herein report two cases of HBV-infected patients with insufficient viral suppression, despite dual antiviral therapy with entecavir (ETV) and tenofovir (TDF). One patient died from aggressive hepatocellular carcinoma (HCC). METHODS: Serum samples from the two patients at different time points were analyzed using ultra-deep pyrosequencing analysis. HBV mutations were identified and transiently transfected into hepatoma cells in vitro using replication-competent HBV vectors, and functionally analyzed. We assessed replication efficacy, resistance to antivirals and potential impact on HBV secretion (viral particles, exosomes). RESULTS: Sequencing analyses revealed the selection of the rtS78T HBV polymerase mutation in both cases that simultaneously creates a premature stop codon at sC69 and thereby deletes almost the entire small HBV surface protein. One of the patients had an additional 261bp deletion in the preS1/S2 region. Functional analyses of the mutations in vitro revealed that the rtS78T/sC69∗ mutation, but not the preS1/S2 deletion, significantly enhanced viral replication and conferred reduced susceptibility to ETV and TDF. The sC69∗ mutation caused truncation of HBs protein, leading to impaired detection by commercial HBsAg assay, without causing intracellular HBsAg retention or affecting HBV secretion. CONCLUSIONS: The rtS78T/sC69∗ HBV mutation, associated with enhanced replication and insufficient response to antiviral treatment, may favor long-term persistence of these isolates. In addition to the increased production of HBV transcripts and the sustained secretion of viral particles in the absence of antigenic domains of S protein, this HBV mutation may predispose patients to carcinogenic effects. LAY SUMMARY: Long-term treatment with antiviral drugs carries the risk of selecting mutations in the hepatitis B virus (HBV). We herein report two cases of patients with insufficient response to dual tenofovir and entecavir therapy. Molecular analyses identified a distinct mutation, rtS78T/sC69∗, that abolishes HBsAg detection, enhances replication, sustains exosome-mediated virion secretion and decreases susceptibility to antivirals, thereby representing a potentially high-risk mutation for HBV-infected individuals.