Background: Clarithromycin is the most commonly suggested antibiotic in Helicobacter pylori eradication regimens. Increasing antibiotic resistance rate to clarithromycin is the main reason for therapeutic failure. The resistance is associated with mutations in the 23S rRNA gene. Aim: The purpose of this study was to determine the clarithromycin resistance rate of isolated H. pylori strains from referral patients in Kashan, Iran. Methods: In total 95 H. pylori strains isolated from gastric mucosa of 246 patients with different clinical signs underwent gastroscopy in Kashan, Iran in 2013. The Epsilometer test was used to determine the minimum inhibitory concentrations (MICs) of clarithromycin (CLR) in isolated H. pylori strains. Occurrence of mutation in 23S rRNA gene of clarithromycin-resistant strains was investigated with polymerase chain reaction-sequencing method. Results: Antibiotic susceptibility pattern showed that 32 strains (33.7%) were resistant to clarithromycin (MICs ≥1 μg/ml), 33.7% were intermediate resistant, while 31 of H. pylori strains (32.6%) were susceptible. The 23S rRNA gene mutations at positions A2143G and A2142G were detected in H. pylori clarithromycin-resistant strains. Conclusion: The resistance rate to clarithromycin in Iran is high. H. pylori clarithromycin-resistant strains were not associated with sex, age, and disease. The A2143G mutation in clarithromycin-resistant H. pylori strains was the predominant finding.