Hepatitis B virus (HBV) strains, resistant to at least two anti-HBV agents from different subclasses of nucleos(t)ide analogues (NUCs) without a cross-resistance profile, are defined as multidrug-resistant. However, there are limited in vivo data for resistance to multiple NUCs. In this study, we report the case of the emergence of a multidrug-resistant HBV strain in a Turkish patient receiving sequential therapy. Polymerase gene mutations of HBV were detected using direct sequencing, line probe assay and clonal analysis. Twelve months after the start of lamivudine (LAM) therapy, virological breakthrough occurred (4.2E+07 IU/ml) and the rtM204V variant was detected in the patient's sera: adefovir (ADV) was added to the therapy. ADV therapy was continued as monotherapy for 11 months, until the occurrence of clinical breakthrough i.e. alanine aminotransferase (ALT) 60 IU/L, and emergence of drug resistance to ADV (rtN236T). At that time, switch therapy was resumed with ADV + entecavir (ETV) in combination for 5 months. In the 18th month of the ETV monotherapy, direct sequencing showed reduced susceptibility to ETV (rtL180M+rtM204V). Currently, ETV + tenofovir (TDF) are being used as antiviral treatment and the HBV DNA load has decreased substantially (<1.0E+02 IU/ml). In conclusion, we have detected an HBV strain with multidrug-resistance, which had a very fast course of development. Patients with a multidrug-resistant profile should be more frequently followed up both by direct sequencing and line probe assay, for the detection of possible novel HBV variants and low level mutants present in the viral population, in case of the sudden emergence of drug resistance.