Objective To establish a convenient,accurate and practical method for detection of adefovir dipivoxil resistance-as- sociated mutation in hepatitis B virusrtA181V/T/S and rtN236T mutations.Methods According to HBV complete sequences in GenBank,two pairs of primers were designed to amplify the region of HBV reverse transcriptase in order to introduce a BglI restriction site upon PCR product of wild type (wt) and a BseDI restriction site upon PCR product of rt236 mutant type.After amplification,the PCR products were digested with BglI and BseDI separately.We used this method to detect wild,rt181 mu- tant,rt236 mutant plasmids and 3 chronic hepatitis Bpatients' serum with obvious ADV resistance-associated mutations.We also tested the sensitivity of this method by mixing the wild and mutant plasmids in different proportions.Results The method could detect rt181 and rt236 mutations simultaneously.The result of RFLPanalysis was in accordance with that of DNA se- quencing and cloninganalysis.This method could detect the mutants even when they comprised only 10% of the total viruspopulation.Conclusions The PCR-RFLPmethod with high sensitivity can detect rt181 and rt236 mutations simultaneously.It can be used for early detection of ADV resistance-associated mutation in hepatitis B virus.