Gastric Helicobacter pylori (H. pylon) plays an important role in the pathogenesis of duodenal ulcer (DU), although not all H. pylori infected persons will develop disease. Duodenal H. pylori was supposed to be one of the factors related with DU. The aim of this study was to investigate whether H. pylori in the duodenum of patients with DU plays a critical role in the pathogenesis of DU regarding the gastric H. pylori status. Furthermore, it was to determine the prevalence of duodenal H. pylori infection in Thailand. Ninety three patients were included in the study. They underwent gastroscopic evaluation for dyspeptic symptoms and none of them had previous H. pylori eradication therapy. An upper gastrointestinal endoscopy was performed and two specimens were collected each form the antrum, midcorpus and duodenal bulb in order to diagnose H. pylori infection. The gold standard for H. pylori detection is a positive specimen culture or polymerase chain reaction (PCR) assay for the vac A gene or positive urease test plus H. pylori seen in the pathology. Ninety three dyspeptic patients (43 males and 50 females; mean age 48.2 years; range 22 to 79 years) were included in the study. Duodenal H. pylori was detected in 31/93 (33.33%) patients which included 15 (48.38%) patients with duodenal ulcer, 2 (6.45%) patients with gastric ulcer and 14 (45.16%) patients with NUD. Five of thirty one (16.21%) patients with duodenal H. pylori infection had negative gastric H. pylori. These five patients included 1 with DU, 1 with DU and 3 with NUD. Duodenal H. pylori was associated with DU dependent of the presence of gastric H. pylori (p<0.05) and there was no association between duodenal H. pylori with negative gastric H. pylori and duodenal ulcer (p>0.05). Duodenal H. pylori is associated with duodenal ulcer dependent on the presence of gastric H. pylori. These results suggest that transmission of gastric H. pylori to the duodenum was prerequisited for the formation of DU.