Background: Helicobacter pylori has been classified as the class I carcinogenic agent by world health organization. Colonization of the human stomach with H. pylori is a risk factor for gastroduodenal diseases. The secreted vacA toxin is an important H. pylori virulence factor that causes multiple alterations in gastric epithelial cells and T cells. Several families of vacA alleles have been described, and H. pylori strains containing certain vacA types (s1 and m1) are associated with an increased risk of gastric disease, compared to strains containing other vacA types (s2 and m2). We examined the association between H. pylori vacA s alleles and gastroduodenal diseases in Iran. Methods: A total of 149 H. pylori strains were obtained from patients with gastritis, peptic ulcer, and gastric cancer referring to endoscopy units of several cities in Iran. Biopsy culture and DNA extraction were performed and the frequency of vacA s alleles was investigated by using PCR amplification. Linear regression and binary logistic regression models were used to analyze the association between vacA (vacuolating cytotoxin A) s alleles and gastroduodenal diseases. Results: There was no significant association between the frequency of vacA s alleles and gastroduodenal diseases (gastritis or peptic ulcer disease and gastric adenocarcinoma (P> 0.05)). Conclusion: It is proposed that the H. pylori vacA s1 genotype could not be considered as an important determinant of gastroduodenal diseases in Iranian population and probably if s1 allele is associated with other virulence alleles of this gene, it will cause diseases.