Background: The Helicobacter pylori (H. pylori) antibiotic resistance pattern differs geographically, knowledge of local antimicrobial resistance pattern is key to successful eradication. The current study was performed to investigate the resistance of H. pylori to 5 commonly used antibiotics in Yangzhou.Methods: A total of 461 H. pylori strains were collected from April 2018 to September 2019 in Yangzhou and tested for their susceptibility to clarithromycin, levofloxacin, metronidazole, amoxicillin, tetracycline using gene chip technology.Results: The resistance rates of H. pylori to clarithromycin, levofloxacin, metronidazole, amoxicillin and tetracycline were 41.0%, 44.9%, 38.8%, 6.3% and 1.1%. In addition, sixteen multiple resistance patterns were detected, and fortunately there were no strains resistant to all five antibiotics. The vacA s1 allele was detected in each strain, 64.0% of the isolates were s1/m2 genotype, 36.0% s1/m1 genotype. In multivariate analysis, history of chronic superficial gastritis was significantly associated with clarithromycin resistance, strains isolated from subjects with gastrointestinal symptoms had a significantly higher likelihood of metronidazole resistance, and antibiotic resistance of H. pylori was independent of both sex and age.Conclusion: The resistance rate of H. pylori to clarithromycin, levofloxacin and metronidazole were very high in Yangzhou, the results will help in selecting effective eradication regimens.