〔Abstract〕 Objective To investigate the phenotype of amoxicillin(AMX) unstable resistantHelicobacter pylori(Hp) evolving into AMX stable high level resistance and the detection of its mutated genes. Methods Using the frozen Hp strain H390 as the starting strain, the clones resistant to AMX were continuously cultured on the medium with increasing AMX concentration, and the minimum inhibitory concentration(MIC) of the resistant clones was detected. After frozen at-80 ℃ for 3 months, the drug resistance was stable according to whether the MIC decreased after frozen storage. Genome sequencing analysis and efflux pump inhibition assay were performed on cloned H390r and parental strain H390 with the highest AMX MIC value, and gene mutations associated with the high level AMX resistance obtained by H390r were detected and identified. Results Four AMX high level resistant clones were obtained by AMX screening with MICs of 12, 32, 64 and ≥256 mg/L, respectively, and none of the MICs were altered after freezing at-80 ℃. Compared to the parental strain H390, the AMX stable resistant clone H390r had mutations in several genes, includinghefCencoding the RND efflux system,hopBandhopCencoding the pore proteins andftsIencoding the penicillin binding protein, which were associated with AMX resistance. H390r was substantially reduced in MIC to AMX in the presence of efflux pump inhibitors. Conclusion AMX can screen stable resistant clones from unstable resistant Hp. H390r had mutations inhefC,hopB,hopC, andftsIassociated with AMX resistance. These mutations may be the main reason why H390r acquired a stable high level of resistance to AMX.