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Objectives Helicobacter pylori gastritis is considered an infectious disease, regardless of symptoms and stage of disease. Most consensus documents recommend empirical therapy based on local antimicrobial susceptibility patterns. We aimed to provide clinically useful information about primary and secondary antimicrobial resistance to antimicrobials commonly prescribed for H. pylori. Methods Overall, 31,406 gastroduodenal biopsies and 2,641 string tests from patients over 15 years of age were plated on selective media, isolating H. pylori in 36.7% of biopsies and 50.7% of string tests. Susceptibility testing could be performed in 96.6% (12,399/12,835) of H. pylori isolates. Polymerase chain reaction (PCR) was also used to detect H. pylori and its resistance to clarithromycin, providing susceptibility data for 112 patients with negative culture results. Results Resistance to amoxicillin and tetracycline was unusual (0.6% and 0.2%, respectively). Rates of primary resistance to clarithromycin and metronidazole remained steady over the 22-year study period, at around 14% for clarithromycin and 30% for metronidazole, while primary resistance to levofloxacin tripled (from 7.6% in 2000 to 21.7% in 2021, P < 0.001) and increased with patient age. Notably, 1.8% of isolates were multiresistant to clarithromycin, metronidazole, and levofloxacin. Overall, secondary resistance rates were higher (P < 0.0001) than primary resistance rates for clarithromycin (42.5% vs 14.1%), metronidazole (40.9% vs 32%), and levofloxacin (21.5% vs 17.1%). Conclusion Determination of susceptibility for H. pylori by culture and/or PCR in patients undergoing endoscopy could facilitate the implementation of tailored therapy and guide the choice of empirical therapy when susceptibility testing cannot be performed, potentially helping limit the emergence of antimicrobial resistance.