Objective To evaluate combined sampling at multiple sites of gastric mucosa for Helicobacter pylori (HP) culture.Methods A total of 258 patients with upper gastrointestinal symptoms received 13C-urea breath test between August 2014 and May 2015.During endoscopy,gastric mucosa biopsy samples from the lesser curvature of the antrum (A),the greater curvature of the antrum (B),gastric angle (C) and the body of the stomach (D) were collected to isolate HP strains.The positive rates of HP based on combined sampling and single site sampling were compared with a Nemenyi test.Results Consistency between 13C-urea breath test and HP culture was 82.56％.There was significant difference between the single site sampling and two-site sampling in the positive rate of HP,except for the body of the stomach (P＜0.05).There was significant difference in the positive rate of HP between the single site sampling and three-site sampling (P ＜ 0.05).There was no significant difference between any two-site sampling in the lesser curvature of the antrum and the body of the stomach,gastric angle and the body of the stomach,the greater curvature of the antrum and the body of the stomach,and any three-site sampling (P＞0.05).Conclusion The combined sampling of the lesser curvature of the antrum and the body of the stomach have the highest cost-effectiveness in HP culture compared with the single site sampling and three-site sampling.