Background: High prevalence and low eradication rates of Helicobacter pylori have become major challenges to clinicians. The growing antibiotic resistance rate via mutational changes is the primary reason for the sharp decline in the H. pylori eradication rate. Aim: To study the role ofgenetic imitations in the antibiotic resistance of H. pylori. Methods: H. pylori strains were isolated between 2017 and 2018 from patients with H. pylori infection. Antimicrobial susceptibility testing was performed for amoxicillin, fora-zolidone, clarithromycin, metronidazole, levofloxacin and tetracycline using the E-test method. Gene mutations were investigated by PCR-RDB assay for the antibiotic-resistant strains. Results: In total, 118 patients with H. pylori infection were enrolled, including 21 subjects from whom strains failed to be isolated. Only 6 strains were sensitive to all of the antibiot-ics tested. Isolates were characterized by no resistance to amoxicillin (0%), furazolidone (0%), and tetracycline (0%); a concerning resistance rate to clarithromycin (27.8%) and levofloxacin (29.9%); and a high resistance rate to metronidazole (88.7%). Of all the clarithromycin-resistant strains, 88.0% had the A2143G mutation in 23S rRNA, while the A2142G mutation was not detected. N87K, D91G, D91N and D91Y were four common mutations in the levofloxacin resistance-determining region of gyrA. The G616A muta-tion (30.2%) was not prevalent in metronidazole-resistant strains. Conclusions: Metronidazole should not be considered when eradicating H. pylori in adults in Zhejiang, China. The first-line empirical regimen for H. pylori eradication should include amoxicillin if the patient is not allergic to it. Gene imitations play an important role in the antibiotic resistance of H. pylori, especially clarithromycin and levofioxacin.