A real-time based amplification assay with molecular beacons was used to detect and quantify PCR amplicons to discriminate between the newly described Lamivudine-resistant YSDD variant, a known YIDD variant and wild-type Hepatitis B virus (HBV) DNA in the YMDD region of the polymerase gene. Using this assay, we retrospectively analysed samples from two HBV chronically infected Asian twin sisters, starting 9 weeks before therapy, during and between two periods of treatment with Lamivudine. In order to analyse more accurately the dynamics of variant DNA loads during and after therapy, this real time assay was compared to three other mutation analysis techniques, restriction fragment length polymorphism (RFLP), InnoLipa HBV-DR assay and direct sequence analysis. With this technique, new information on the dynamics of variants during and after therapy was obtained.