[Objective] To detect HBV YMDD motif mutants using RDB hybridization assay in lamivudine treated patients with chronic hepatitis B virus infection,as well as to evaluate the detection capability for clinical application.[Method] HBV DNA was extracted from serum for a total of 242 cases,after the PCR amplification,the hybridization was performed.By comparing the RDB assay results to sequence analysis,the concordant results were analyzed.The sensitivity and detection capability for mixed infection samples are also evaluated.[Results] There are 236 of concordant results for RDB assay and sequencing were obtained in a total of 242 cases,accounting for 97.5%.For all of the cases,there are 58 cases with coexisting mutant viruses in wild type viruses,accounting for 24%.The sensitivity of RDB hybridization assay for HBV YMDD motif mutants was 103 IU/mL,and approximately 10% mutant type strains can be detected from a mixed infection sample.[Conclusion] The RDB hybridization assay for HBV YMDD motif mutants is a simple,accurate,and economic method and it may be a promising tool for clinical application.