Helicobacter pylori is a Gram-negative, microaerophilic, and spiral-shaped bacterium. H. pylori infection is most common bacterial infection in the world whose main reservoir is the human stomach. Long-term H. pylori infection results in chronic gastritis, and intestinal metaplasia of the gastric mucosa that represent significant risk factors for gastric cancer. In this study, a total of 60 Gastric biopsy samples were collected in BHI and were inoculated on Columbia blood agar supplemented with DENT. Microscopy and biochemical test were performed to identify H. pylori. The susceptibility profile of H. pylori isolates were evaluated using Kirby Bauer disk diffusion method. The DNA of selected isolates were isolated using phenol/chloroform method. After amplification of 16s rRNA, PCR was done for the detection of resistance genes including Metronidazole resistance (rdxA gene), Clarithromycin resistance (23s rRNA) and Amoxicillin resistance (pbp1A gene). In a total of 80 samples, H. pylori was isolated from 58 samples showing percentage of 72.5%. Among the positive patients, there were 62% (n=36) of female and 38% (n=22) male positive patient. H. pylori was more common in age between 30-50 years n=32 (55.17%). Most of H. pylori isolates shown resistance to Metronidazole 90% (n=36), intermediate resistance to Clarithromycin 57.5% (n=23) and Amoxicillin 47.5% (n= 19) and susceptible to Levofloxacin 65% (n=26). A total of 20 isolates were selected for the detection of resistance genes. In this total, 13 were positive for Metronidazole resistance gene, 10 were positive for Amoxicillin and 7 were positive for Clarithromycin resistance gene