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Aims: We described the presence of Helicobacter pylori (HP) and estimated the prevalence of primary and secondary resistance using molecular detection in gastric biopsies of Ecuadorian patients. Methods and Results: 66.7% (238/357) of the patients demonstrated the presence of HP using CerTest qPCR. Of these, 69.79% (104/149) were without previous HP eradication treatment and 64.42% (134/208) with prior HP eradication treatment. The mutation‐associated resistance rate for clarithromycin was 33.64% (primary resistance) and 32.82% (secondary resistance), whereas that in levofloxacin the primary and secondary resistance was 37.38% and 42%, respectively. For tetracycline and rifabutin, primary and secondary resistance was 0%. Primary and secondary resistance for metronidazole and amoxicillin could not be evaluated by genotypic methods (PCR and sequencing). Conclusions: The analysis of mutations in gyrA, 23S rRNA and 16S rRNA is useful to detect bacterial resistance as a guide for eradication therapy following failure of the first‐line regimen. Significance and Impact of the Study: This study carried out in an Ecuadorian population indicates that the resistance of HP to first‐line antibiotics is high, which may contribute to the high rates of treatment failure, and other treatment alternatives should be considered. [ABSTRACT FROM AUTHOR]