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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., (© 2021 The Society for Applied Microbiology.)