BACKGROUND: Treatments for Helicobacter pylori (H. pylori) eradication include the use of antibiotics and a proton-pump inhibitor. Antibiotic resistance is a major concern for two drugs: levofloxacin and clarithromycin. The aim was to determine the prevalence of levofloxacin resistance (LevoR) and clarithromycin resistance (ClaR) in an urban population in Santiago, Chile. METHODS: Gastric mucosa biopsies were obtained for DNA isolation from 143 H. pylori-positive individuals aged 18-80 years. Direct sequencing of the quinolone-resistance determining region (QRDR) of the gyrA gene was used to determine LevoR. ClaR was determined using restriction-fragment length polymorphism or 5'exonuclease assay. RESULTS: The prevalences of LevoR and ClaR were 29 and 27%, respectively. LevoR was higher in women than in men (39 vs. 13%, p <0.001), while no sex difference was observed for ClaR (p = 0.123). The prevalence of LevoR increased with age (p-trend = 0.004) but not for ClaR (p-trend = 0.054). In sex-stratified analyses, both LevoR and ClaR increased with age only among women. Older women (>50 years) had a higher probability to carry LevoR strains as compared to men. The prevalence of dual LevoR and ClaR was 12.6%. CONCLUSIONS: The prevalence of ClaR and LevoR is high in Santiago, according to International guidelines that recommend avoiding schemes with antibiotic resistance >15%. Our findings provide evidence to re-evaluate current therapies and guide empirical first- and second-line eradication treatments in Chile.