The treatment of infection is complicated by antibiotic resistance. A high levofloxacin (LVX) resistance rate was previously demonstrated in isolates from gastric mucosa (40%) and esophagus (19%) in individual hosts of a Venezuelan population. We aimed to assess the molecular mechanisms of LVX resistance and susceptibility in isolates from the gastroesophageal mucosa, by studying point mutations in the quinolone resistance-determining region of and genes. Sequencing of and genes ( = 120) helped to identify point mutations in 60 isolates (30 from antrum and 30 from esophagus) of five dyspeptic patients. Double (Asn87Thr and Asp91Asn) and single (Asn87Ile or Asn87Thr) mutations in the gene were identified in the esophageal mucosa. These mutations have been commonly found in the stomach. Occurrence of a single (Asn87Ile) mutation was associated with high resistance (minimum inhibitory concentration ≥ 32 μg/mL) to LVX. Only a single (Ser479Gly) mutation was found in the gene in both mucosae. One patient presented isolates with no mutations in the two genes studied. Isolates with the same mutation pattern in individual hosts revealed identical genetic profiles for these genes, confirming that isolates identified in the esophageal mucosa come from isolates colonizing the stomach. resistance to LVX in the esophagus is related to double- and single-point mutations in and genes, such as those found in the stomach. Levofloxacin should be applied with caution, because its antibiotic effect on is decreasing in Latin America, perhaps owing to high prescription rates.