OBJECTIVE: To establish PCR based detection assays for dup A, ice A, hom B genes of H. Pylori and detect the presence of these genes in Pakistani H. Pylori isolates, and to examine other confounding factors beside dup A, ice A, hom B such as age, gender, diet and lifestyle characteristics with association of H. Pylori-related diseases METHODOLOGY: This cross-sectional analytical study was conducted at gastroenterology Department, Shaikh Zayed Hospital, Lahore and molecular techniques were performed in Centre of Excellence and Molecular Biology, University of Punjab, Lahore, with duration of two years. All the cases with symptomatic dyspeptic patients either gender and with age more than 18 years were included in the study. All the subjected patients were undergone to endoscopic examination. Upper gastrointestinal endoscopy was carried out and specimens were obtained for the gastric biopsy. Specimens were inoculated in the CLO or rapid urease test gel and rest were stored in eppendorf tubes containing normal saline at -20C until DNA extraction. DNA was extracted of the H-pylori by using Gentra DNA extraction Kit (Life Technologies, USA) according to instructions given in protocol of the kit for Qualitative PCR detection of DNA of H. Pylori. The PCT primers sets were designed for the specific detection of dup A, ice A and hom B gene of H. Pylori using primer 3 software. The designed PCR primer sets specific for the H. Pylori dup A, ice A and hom B genes were used for PCR amplification. PCR was performed for the amplification of dup A, ice A and hom B genes. The desired genes were quantified using Real-time PCR. The PCR bands of the amplified genes were gel eluted and were stored at minus 20 degree C till to be used for sequence analysis. RESULTS: Mean age of the cases was found 41.22+8.04 years. Male were more affected 118 (60.2%) as compare to female 78(39.8%). Epigastric discomfort was found in the 196(100%)of the cases where as Bloating and Belching cases were noted as 180(91.8%) while Nausea a & vomiting cases as observed as 138(70.4%). The life style distribution exhibited that Hand wash 188(95.9%)was observed most frequent in all cases and exercise 36(18.4%) of cases, however municipality source water was more common following by Fresh water 48(24.5%) and Filtered water as 4(2.0%) of cases. As indicated by this study CLO/rapid urease test as well as Polymerase chain reaction quantity was noticeably 100% positive in all cases. As per H.pylori genes distribution Hom B was the most common 76(38.8%). A significant association was found in the gene distribution of the H. pylori among both genders male were more linked to Dup A, while female were mostly associated with Ice A and Hom A genes, P-value 0.001. Different type of diets is significantly associated with h pylori genes P value 0.001. Water bad sources were highly associated with h pylori genes as genes were mostly found in municipality water as compare to filtered and fresh water p- value 0.001. CONCLUSION: prevalence of hom-B gene most frequent as compare to Dup A and ice A genes, in the patients having dyspepsia. These genes were strongly associated with bad life style and bad sewerage system along with some poor dietary habits.