Objective:To construct a new direct amplification PCR method to detect HBV rtA181T drug resistant mutation by nested PCR and specific primers. Methods:Nested PCR was performed to amplify the HBV polymerase rt domain fragment in the first round, and the base in the 3′end of the sense primer was designed same as the mutation one of HBV DNA rtA181T in the second round. Thus, the amplified fragment was the HBV DNA rtA181T mutation fragment. Using this method, we examined 43 specimens of various HBV DNA loading with HBV DNA rtA181T mutation and analyzed the sensitivity of this method; and compared it with the PCR-Sanger sequencing method in consistency under the level of low or high copy DNA HBV. Results:Confirmed by PCR products sequencing, this nested PCR based specific primers had successfully detected the HBV DNA rtA181T drug resistant mutation. This method had confirmed rtA181T drug resistant mutation even when the virus load was 24 U/-滋L and the mutation strain was less than 10% in the whole quasi-species pool. By detection of the 43 specimens, we found that the sensitivity of this method was 100%, significantly superior to Sanger sequencing, which was 74%(P < 0.05). Conclusion:Based on nested PCR and specific primers, our new method can successfully detect HBV DNA rtA181T mutation. Especially in low virus load circumstance, this new method is superior to Sanger sequencing in sensitivity, and also shows good specificity, thus is beneficial to early detection of HBV DNA drug resistant mutation and important for adjustment of the anti-HBV therapy in time.