show Abstracthide AbstractStrategies to increase the production rate of chicken alter the natural process of microbial colonisation and nutritional performance. Studies have focused on understanding the diversity, composition, and gene content of gut microbiota in chickens to improve the management of such affections. However, the lack of sufficient reference genomes and genes, limits the interpretation of sequencing data and restrain deeper studies of detailed functions, particularly on less explored anatomical regions such as crop and small intestine. In this study, 696 strains obtained from crop, jejunum and ileum of chicken were isolated and genome analysed in order to observe their metabolic profiles, colonisation preferences and adaptive strategies on these regions, and to serve as future references. Among the collection, nine isolates represented new species and expanded our knowledge of the specialised functions of gut chicken microbiome. These strains were consistently found within the upper regions (crop to ileum). Strains of Limosilactobacillus were found to be more abundant at crop while Ligilactobacillus strains dominated ileum. Strains originated from crop presented a high number of glycosidases specialised in complex polysaccharides as for jejunum and ileum, strains presented higher number of molecules that interact to the host such as collagenases and hyaluronidases. Such differences indicated a preferential persistence of species and their adaptations along the tube. These results represent the first deep approach of bacteria from the upper digestive system of chicken and describes the unexplored taxonomic and functional diversity of bacteria inhabiting these regions improving the potential handling of chicken microbiota with biotechnological applications.