Background. Helicobacter pylori (H. pylori) resistance to clarithromycin is increasing worldwide. Data on the prevalence of H. pylori resistance are limited in Tunisia. Gap statement. Given that H. pylori resistance to clarithromycin has not been studied in Tunisia since 2010, there was a need to determinate its prevalence and the principal mutations implicated in this resistance. Aim. The aims were to define the prevalence of H. pylori infection among symptomatic patients and to determinate the level of clarithromycin resistance among these patients and the main mutations conferring this resistance. Methods. We conducted a cross-sectional study from March 2017 to February 2020 in the Hepato-Gastroenterology Department of Hedi Chaker University Hospital in Sfax that included 124 Tunisian patients who underwent gastroduodenal endoscopy with biopsies. Mutations conferring resistance to clarithromycin were detected using the Allplex H. pylori and ClariR PCR Assay. Results. Out of 124 biopsies, 101 (81.5 2 %) were PCR-positive for H. pylori . Mutations conferring resistance to clarithromycin were detected in 30/95 (31.6 %) of patients. The rate of primary resistance was 25.3 % and of secondary resistance 62.5 %. The most frequently detected mutation was A2143G (86, 90%) followed by A2142G (11, 36%). Seven patients had a double mutation A2143G–A2142G. The factors independently associated with resistance to clarithromycin were diabetes, high blood pressure, the presence of a bulbar ulcer on endoscopy and the presence of gastric atrophy on histology. Conclusion. Detection of more than 25 % of strains with clarithromycin resistance mutations makes the H. pylori first-line treatment with clarithromycin questionable in our setting, and a review of empirical treatment of H. pylori is urgently needed.