Long term antiviral therapy with nucleos(t)ide analogs (NAs) may lead to the emergence of drug-resistance viral mutants in chronic hepatitis B virus (HBV) patient. The purpose of this study was to identify adefovir dipivoxil (ADV) resistance mutations of HBV polymerase and determine effective drugs to replace ADV. The reverse transcriptase (RT) coding region was PCR-amplified using HBV DNA extracted from patient blood samples and sequenced. Nineteen substitution mutations were detected. Among them, rtN238R, rtT240Y and rtN248H were often observed in patients receiving ADV administration. These three potential drug resistant sites were introduced into HBV replication-competent plasmids. The in vitro susceptibility of both wild-type (WT) and mutant-type (MT) HBV to NAs was analyzed by Southern blotting and quantitative real-time PCR. The rtN238R, rtT240Y and rtN248H substitutions had no obvious effect on HBV DNA replication or gene expression. The in vitro susceptibility analysis showed that rtN238R, rtT240Y and rtN248H substitutions were responsible for the reduced susceptibility to ADV, and demonstrated a 5.42-, 2.89- and 5.72-fold increase in resistance towards ADV, respectively. However, HBV harbored these mutations retained normal susceptibility to LMV, LdT, ETV and TDF.