The nucleotide analog adefovir dipivoxil (ADV) is an effective antiviral treatment for chronic hepatitis B virus (HBV) infection, with resistance to ADV estimated to occur less frequently than resistance to lamivudine treatment. The detection of ADV resistance mutations is necessary during therapy to monitor and anticipate possible treatment failure. The INNO-LiPA HBV DR v2 (LiPA; Innogenetics, Ghent, Belgium) is a DNA hybridization line probe assay for the detection of HBV polymerase mutations associated with resistance to lamivudine and ADV. Evaluation of this assay to detect ADV resistance mutations was performed by analyzing 38 patients treated with ADV. Serial samples taken at 6-month intervals during treatment were available for most patients. A total of 124 samples were analyzed by both LiPA and sequencing. By LiPA analysis, 12 patients (31.5%) were found to have mutations associated with resistance to ADV (rtA181V/T and/or rtN236T). This contrasted with sequence analysis, which found nine patients (24%) with either or both mutations. Twice as many samples were rtN236T positive by LiPA (18 of 124) compared to sequence analysis (9 of 124). LiPA detected the rtN236T mutation at least 6 months earlier than its detection by sequencing in patients for whom consecutive serum samples were available. Although less sensitive, sequencing has the advantage of providing information on other polymerase mutations not represented on LiPA strips. The INNO-LiPA HBV DR v2 assay is a very sensitive and specific assay for the detection of the rtN236T mutation associated with resistance to ADV.