Background: Helicobacter pylori is an important human pathogen linked with severe gastrointestinal (GI) problems. This Gram-negative microaerophilic bacteria, classified as a Class 1 carcinogen (World Health Organization 1994), infects over half of the world’s population. In India, particularly in Southern India, gastric cancer is the most devastating consequence of H. pylori infection. The present study was conducted to evaluate the prevalence of H. pylori in patients with upper GI (UGI) disorders by phenotypic and genotypic methods and to identify the presence of cancer-inducing cytotoxin-associated-gene A (CagA) gene from isolated H. pylori. Methods: Patients with symptomatic GI disorders undergoing UGI endoscopy were included. Four bits of gastric biopsy samples in brain - heart infusion bottle will be received from medical gastroenterologist division from each patient for microbiological and molecular analysis. The identification of H. pylori was done by conventional methods using Skirrow Campylobacter Media and its growth supplements and molecular methods by the polymerase chain reaction (PCR). DNA extraction performed by using Amp ready reagent and kept at -20°C till the processing of samples. Data collection was done using a pro forma and analysis of data was done and expressed as frequency and percentage. Results: A total of 102 gastric biopsy samples obtained from medical gastroenterology department were processed by conventional and molecular methods. Out of these, only 7 (6.8%) were found to be culture positive, 24 (23.5%) were positive for rapid urease test, but 38 (37.25%) isolates were found to be positive for H. pylori by PCR. These isolated H. pylori strains were further processed for identifying cancer-inducing cagA gene by molecular methods, out of 38 isolates 23 (60.5%) were found to be positive for CagA. Conclusion: Using PCR to detect H. pylori and the virulent cagA gene directly from a biopsy is a rapid, reliable, and better alternative to other conventional methods. © 2023 Elsevier B.V., All rights reserved.