Objective and background: It is well document Helicobacter pylori, has been a major causes of peptic ulcer, gastric ulcer, duodenal ulcer disease and is an early risk factor for gastric carcinoma. This study has been undertaken forisolation of  Helicobacter pylori form clinical specimens using culture technique and detection the role of this technique in the investigation of H. pylori infection. Also detection of anti -H. pylori IgG using enzyme linked immune sorbent assay (ELISA), further molecular detection of H. pylori genome by amplification of 16s r-ribonucleic acid gene with 520 pb by polymerase chain reaction PCR. Patients and Methods: Atotal of Eighty five adult patient attending the gastro endoscopy unit of Al- Ramadi Teaching Hospital to undergo selective esophageal gastroduodenoscopy (OGD) were studied. They were all suffering from clinical manifestation of duodenal ulcer (DU), gastric ulcer (GU), peptic ulcer (PU) and non-ulcer dyspepsia (NUD). Culture and ELISA technique were used. Further molecular detect of 16 s r RNA gene was performed by PCR.  Results: A total 7(8.23%) with GU, 5 (5.91%) with PU, 10 (11.75%) with DU and 28 (32.94%) patients with NUD were positive by urease test (UT). A total 3 (3.52%) patients with GU, 4 (4.71%) patients with PU and 3 (3.52%) patients with DU were positive by culture while no one of the patients of NUD was positive by culture. By using ELISA technique 18 (21.2%) patients were positive by ELISA. In the present study, the presence of Helicobacter DNA was investigated using a Helicobacter species-specific 16srRNA PCR amplification.DNA extracted from human blood and bacterial colonies. PCR showed 20(100%) of cases positive result from human blood while 6 (60%) of cases were positive result of PCR from bacterial colonies. Conclusions: The study concludedthat urease test was useful as preliminary screening test and important in give indicate for H. pylori presence. Further, the role of culture was very important in the detection of H. pylori in clinical samples. Furthermore, the immunologically bases serological test, detection of anti-H pyloriIgG by ELISA technique was undependable serological test. It can be used as preliminary screening test for detection of H. pylori in association with the other tests. FurtherPCR was sensitive and specific test for diagnosis of H. pyloriinfection.