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Abstract The gut microbiome is influenced by host species and the environment, but how the environment influences the microbiome of animals introduced into a new ecosystem has rarely been investigated. Freshwater mussels are aquatic fauna, with some threatened or endangered species propagated in hatcheries and introduced into natural systems as part of conservation efforts. The effects of the environment on the freshwater mussel gut microbiome were assessed for two hatchery-propagated species (Lampsilis ovata, Lampsilis ornata) introduced into rivers within their natural range. Mussels were placed in rivers for 8 weeks, after which one subset was collected, another subset remained in that river, and a third subset was reciprocally transplanted to another river in the same river basin for a further 8 weeks. Gut microbiome composition and diversity were characterized for all mussels. After the initial 8 weeks, mussels showed increased gut bacterial species richness and distinct community composition compared to hatchery mussels, but gut microbiome diversity then decreased for mussels that remained in the same river for all 16 weeks. The gut bacterial community of mussels transplanted between rivers shifted to resemble that of mussels placed initially into the recipient river and that remained there for the whole study. All mussels showed high proportions of Firmicutes in their gut microbiome after 8 weeks, suggesting an essential role of this phylum in the gut of Lampsilis species. These findings show that the mussel gut microbiome shifts in response to new environments and provide insights into conservation strategies that involve species reintroductions.