The duodenum represents a unique niche within the digestive tract, characterised by the presence of aggressive digestive enzymes and bile.  Due to the harsh conditions, the duodenum has long been considered sterile, with bacteria only present due to cross contamination or bacterial overgrowth due to immune deficiencies or motility disorders.  Emerging evidence from molecular and culture based studies supports the presence of bacteria in this region in healthy individuals.  However, these studies are constrained by issues of contamination during sampling, particularly from the upper gastrointestinal tract (oral cavity, saliva and stomach).  In order to overcome this, we have utilised biopsy forceps protected by a sheath, allowing targeted, aseptic sampling of the duodenal mucosa.  Biopsy samples were collected using the aseptic device from individuals undergoing upper gastrointestinal endoscopy with ethical approval.  Following gDNA extraction, amplicon libraries spanning the V6-V8 region of the 16S rRNA gene were constructed, sequenced using the Illumina MiSeq platform, and analysed through QIIME.  Microbial DNA was detected in all samples, indicating the presence of a duodenal mucosal microbiota.  Sequence analysis revealed a community dominated by Streptococcus, representing up to 50% of the total bacterial load.  The other most abundant genera identified were Prevotella, Lactobacillus, Veillonella, Neisseria and Porphyromonas.  While these genera are typically identified in the oral microbiota, these results indicate they are also located in the duodenal mucosa.  Concurrent sequencing of reagent-only controls revealed the presence of a “microbiota” in common laboratory reagents, in line with recent studies.  However, there was relatively limited overlap between OTUs identified in reagents and duodenal samples (primarily Pseudomonas and Caulobacteraceae).  The bacteria identified in this study therefore represent members of a unique duodenal mucosa-associated microbiota.  Further investigation is ongoing into the adaptation of this bacterial community to the duodenal niche and links between the duodenal microbiota and disordered gut function.