Objective(s): RESISTANCE to antimicrobial agents, particularly metronidazole and clarithromycin, is frequently observed in Helicobacter pylori and may be associated with treatment failure. This RESISTANCE rate varies according to the population studied. The aim of this study was to assess the pattern of antimicrobial RESISTANCE of H. pylori isolates from dyspeptic patients in Isfahan.Materials and Methods: Antral gastric biopsies from 230 dyspeptic patients were cultured. Susceptibility testing to commonly used antibiotics performed on pure cultures of 80 H. pylori-positive isolates by Modified Disk Diffusion Method (MDDM). Genomic DNA extracted and subjected for study of entire genomic pattern using Random Amplified Polymorphic DNA- Polymerase Chain Reaction (RAPD-PCR). Results: The overall rates of primary RESISTANCE were 30.0%, 8.75%, 6.25%, 3.75%, 3.75%, and 2.50% for metronidazole, ciprofloxacin, clarithromycin, azithromycin, tetracycline, and amoxicillin, respectively. Multiple antibiotic RESISTANCEs were observed in 8 of 27 resistant isolates (29.6%) that mainly were double RESISTANCE with the prevalence of 6.25%. No association between antimicrobial RESISTANCE and either the gender, age or clinical presentation of the patients were detected. In RAPD-PCR, great diversity observed in 27 resistant strains isolated from different patients and this heterogeneity was not significantly different from susceptible strains.Conclusion: Primary H. pylori RESISTANCE to metronidazole in our population was lower than the developing world and even other parts of Iran, to ciprofloxacin was considerable in comparison with results in most other countries. Moreover, antibiotic RESISTANCE had no effect on genomic pattern of H. pylori isolates. Finally, pretreatment H. pylori isolates susceptibility testing is highly recommended.