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Introduction: Helicobacter pylori is a micro-aerophilic gram-negative bacteria that is colonized in the gastric mucosa and duodenum using its own factors such as urease, catalase and lipase. While the stomach is considered the main source of it, other tissues such as the Walder ring, gallbladder, gums, oral lesions and dental plaque have also been suggested as potential sources. Recent studies suggest that adenotonsillar tissue is susceptible to Helicobacter pylori colonization, but the results are very contradictory. Therefore, in the present study, we tried to evaluate the presence of this bacterium in this tissue. Materials and Methods: In this cross-sectional study, which was conducted from October 2017 to October 2016, cases of adenotonsillectomy surgery without a history of antibiotics and bismuth in the last two weeks were included in the study. 2 to 5 mm of samples were removed and placed in a filled vial up to half of the urease test kit solution. Change of color of the solution from yellow to purple was a positive result and no color change after a maximum of one hour, a negative result was recorded. Results: A total of 114 samples were obtained from 64 patients, of which 63 were tonsil samples and 51 were adenoids. In 5 samples (all tissues removed from the tonsil), the urease test was positive (4.4%) and the other 109 samples (95.6%) were negative. There was no significant relationship between urease test result with tissue type (P = 0.063), reason for surgery (P = 0.65) and age (P = 0.07). Conclusion: Our results showed that Helicobacter pylori colonization in adenotonsillar tissue had a frequency of 4.4%, in other words, 5 out of 64 patients (7.8%) had tonsil colonization with Helicobacter pylori, which showed a small amount.