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The gut microbiome is a highly intricate ecosystem that exerts a pivotal influence on the host's physiology. Characterizing fish microbiomes is critical to understanding fish physiology and health, but little is known about the ecology and colonization dynamics of microorganisms inhabiting fish species. In this study, we investigated the bacterial communities of two small-bodied fish species, Cyprinella lutrensis (red shiner) and Notropis stramineus (sand shiner), two fish species where gut microbiomes have not been investigated previously and surrounding waters, collected from rivers in Nebraska, USA. Our study focused on evaluating microbial diversity in small-bodied fish and identifying autochthonous microbes present within these species irrespective of location to better understand bacterial community composition and possible roles of such bacterial species. Our results revealed that both red shiner and sand shiner exhibited gut bacterial communities dominated by typical bacterial phyla found in freshwater fish. The phylum Bacteroidota was minimally abundant in both species and significantly lower in relative abundance compared to the surrounding water microbial community. Furthermore, we found that the gut microbiomes of red shiner and sand shiner differed from the microbial community in the surrounding water, suggesting that these fish species contain host-associated bacterial species that may provide benefits to the host such as nutrient digestion and colonization resistance of environmental pathogens. The fish gut bacterial communities were sensitive to environmental conditions such as turbidity, dissolved oxygen, temperature, and total nitrogen. Our findings also show bacterial community differences between fish species; although they shared notable similarities in bacterial taxa at phyla level composition, ASV level analysis of bacterial taxa displayed compositional differences. These findings contribute to a better understanding of the gut bacterial composition of wild, freshwater, small-bodied fish and highlight the influence of intrinsic (host) and environmental factors on shaping the bacterial composition., (© 2024. The Author(s).)