Objective：To establish a high-throughput method which can rapidly detect Lamivudine-resistant mutation against hepati－tis B virus（HBV） to guide clinical medication. Methods：Serum samples of 150 patients with chronic hepatitis B（CHB），whose quan－titative HBV DNA results were positive（copy number ≥103），were determined by pyrosequencing for detecting Lamivudine-resistantly mutational site via PyroMark ID genetic analysis systems. In order to prepare single-stranded template of pyrosequencing，samples were amplified by nested PCR and were separated by magnetic bead and amplification primer of the polymerase in P region was designed. Results：HBV P region products were attainted by amplified PCR and mutation frequencies of pyrosequencing were characterized by high sensitivity，specificity，stability and were in correlation with Lamivudine-treatment time. With the increase of Lamivudine treatment time，mutation frequencies and drug resistance were increased. Conclusions：High-throughput pyrosequencing may meet the needs of clinical drug testing and design of treatment protocols，which can detect Lamivudine-resistant mutation of HBV in patients treated with Lamivudine．