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Objectives: Drug resistance of Helicobacter pylori is a major clinical problem worldwide. The objective of the present study was to investigate the prevalence of antibiotic-resistant H. pylori in the city of Shenzhen in China, as well as to identify the genetic mutations specifically associated with drug resistance rather than unrelated phylogenetic signals., Methods: Antibiotic susceptibility testing was performed on 238 clinical strains successfully isolated from H. pylori-positive dyspeptic patients who underwent gastroscopy at the Department of Gastroenterology in Shenzhen People's Second Hospital. Following WGS of all strains using Illumina technology, mutation and phylogenetic analyses were performed., Results: The resistance rates were 84.9%, 35.3%, 25.2% and 2.1% for metronidazole, clarithromycin, ciprofloxacin and rifampicin, respectively. An A2143G conversion in the 23S rRNA gene was the primary mutation observed in clarithromycin-resistant strains, whilst N87K/I and D91G/N/Y in GyrA were detected in ciprofloxacin-resistant strains. In RdxA, our results demonstrated that only R16H/C and M21A are significant contributors to metronidazole resistance; there were 15 other sites, but these are phylogenetically related and thus unrelated to metronidazole resistance., Conclusions: There is a high prevalence of metronidazole, clarithromycin and ciprofloxacin resistance and a low prevalence of rifampicin resistance in H. pylori from Shenzhen, China. Omission of phylogenetically related sites will help to improve identification of sites genuinely related to antibiotic resistance in H. pylori and, we believe, other species., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)