Background:The initial intestinal microbiota acquired from different sources has profound impacts on animal health and productivity. In modern poultry production practices, the source(s) of the establishing microbes and their overall contribution during development of gastrointestinal tract communities are still unclear. Using fertilized eggs from two independent sources, we assessed the impact of eggshell-and environmental-associated microbial communities on the successional processes and bacterial community structure throughout the intestinal tract of chickens for up to 6 weeks post-hatch. Results:Culturing and sequencing techniques identified a viable, highly diverse population of anaerobic bacteria on the eggshell. The jejunal, ileal, and cecal microbial communities for the egg-only, environment-only, and conventionally raised birds generally displayed similar successional patterns characterized by increasing community richness and evenness over time, with strains of Enterococcus, Romboutsia, and unclassified Lachnospiraceae abundant for all three input groups in both trials. Bacterial community structures differed significantly based on trial and microbiota input with the exception of the egg-exposed and conventional birds in the jejunum at week 1 and the ileum at week 6. Cecal community structures were different based on trial and microbiota input source, and cecal short-chain fatty acid profiles at week 6 highlighted functional differences as well. Conclusion:We identified distinct intestinal microbial communities and differing cecal short-chain fatty acid profiles between birds exposed to the microbiota associated with either the eggshell or environment, and those of conventionally hatched birds. Our data suggest the eggshell plays an appreciable role in the development of the chicken intestinal microbiota, especially in the jejunum and ileum where the community structure of the eggshell-only birds was similar to the structure of conventionally hatched birds. Our data identify a complex interplay between the eggshell and environmental microbiota during establishment and succession within the chicken gut. Further studies should explore the ability of eggshell-and environment-derived microbes to shape the dynamics of succession and how these communities can be targeted through interventions to promote gut health and mitigate food-borne pathogen colonization in poultry.