The amino acid substitutions rtP177G and rtF249A in the reverse transcriptase domain of hepatitis B virus polymerase reduce the susceptibility to tenofovir

► Potential HBV TDF-resistant sites were selected based on bioinformatics analysis. ► Replication capacity and resistance phenotype were analyzed in vitro and in vivo. ► Two HBV mutants (rtP177G and rtF249A) showed enhanced resistance to TDF. ► The resistant mechanisms are discussed based on the RTs structural comparison.