Background: The aim of the study was to assess the knowledge and practices of primary care physicians in diagnosis and management of Helicobacter pylori (H. pylori) infection in developing country. Methods: This convenient sample based, cross sectional study was conducted in primary care physicians of Karachi, Pakistan from March 2008 to August 2008 through a pretested self-designed questionnaire, which contained 11 items pertaining to H. pylori route of transmission, diagnosis, indication for testing, treatment options, follow up and source of information. Results: Out of 509 primary care physicians, 451 consented to participate with the response rate of 88.6%. Responses of 426 primary care physicians were analyzed after excluding 19 physicians. 78% of the physicians thought that contaminated water was the source of spread of infection, dyspepsia was the most frequent indication for investigating H. pylori infection (67% of the physicians), while 43% physicians were of the view that serology was the most appropriate test to diagnose active H. pylori infection. 77% of physicians thought that gastric ulcer was the most compelling indication for treatment, 61% physicians preferred Clarithromycin based triple therapy for 7-14 days. 57% of the physicians would confirm H. pylori eradication after treatment in selected patients and 47% physicians preferred serological testing for follow-up. In case of treatment failure, only 36% of the physicians were in favor of gastroenterologist referral. Conclusion: The primary care physicians in this study lacked in knowledge regarding management of H. pylori infection. Internationally published guidelines and World gastroenterology organization (WGO) practice guideline on H. pylori for developing countries have little impact on current practices of primary care physicians. We recommend more teaching programs, continuous medical education activities regarding H. pylori infection.