Retrospective analysis of our local HBV reverse transcriptase (rt) sequence database including 973 sequences recovered from 616 patients identified one unique HBV DNA sequence harbouring amino acid (aa) substitution rtA194T, which has been suspected to confer reduced susceptibility to tenofovir but whose implication in antiviral resistance has not been confirmed. This sequence has been recovered from the blood sample of a 35-year-old man presenting with chronic hepatitis B and cirrhosis, at time of initiation of HBV therapy with adefovir (ADV). Retrospective analysis showed that viruses were rt194A eleven months earlier. Nonetheless, rt sequences recovered from the two sequential serum samples showed 98% nucleotide identity and were clustered in phylogenetic reconstruction. Clonal sequencing was performed retrospectively, which showed that rt194A HBV sequences were the only detected in the earliest sample and rt194T HBV sequences were the only detected in the later sample. HBV rtA194T mutants were still the majoritary quasi-species 17 months after being identified for the first time. HBV genotype determined by means of population sequencing then phylogeny reconstruction was E. HBV harboring a rt194T can be naturally observed, although very rarely, in absence of any prior therapy. Indeed, they represent six (0.2%) of three 110 sequences recovered from drug naive patients in the Stanford HBV sequence database (http://hivdb.stanford.edu/HBV/DB/cgi-bin/MutPrevByGenot ypeRxHBV.cgi). In the present observation, we cannot interpret the virological response under anti-HBV therapy due to short follow-up and nonoptimal drug compliance, as indicated by patient’s interview and TDF plasma Ctrough determination.