Background: Helicobacter pylori prevalence and gastric cancer rates are remarkably high in Peru. Effective antimicrobial regimens are essential for successful H. pylori eradication. We aimed at assessing antimicrobial resistance rates to first- and second-line therapeutic agents in H. pylori strains detected in gastric biopsy samples. Materials and Methods: Gastric biopsy samples (antrum and corpus) were collected from therapy-naive patients (n = 154). H. pylori presence in the samples was confirmed by histopathology. Genotypic resistance to clarithromycin and quinolones was determined by real-time PCR. Results: Histology results were 100% concordant with PCR results (97/154; 63% H. pylori-positive in both). In 6% (6/97) of the patients, we found discordant results of H. pylori infection in antrum and corpus samples from the same patient. Resistance rates to clarithromycin and quinolone were 34% (33/97) and 68% (56/82), respectively. Antimicrobial resistance to both antimicrobials was 30% (25/82). Conclusion: Antimicrobial resistance rates of H. pylori to clarithromycin and quinolones are very high in Lima, Peru. Many first- and second-line, empiric eradication regimens may not be recommended for Peruvian patients.