Freshwater mussels have a unique life history in which larval mussels (glochidia) act as obligate parasites to fish hosts. Host selectivity may be species specific, and identification of host fish is a critical step in conservation planning for individual mussel species. The Flint River harbors ~23% of the freshwater mussel (order Unionida) diversity in the state of Georgia, USA. Nine species in the basin are state or federally listed, and local diversity is threatened by shifting hydrologic conditions, increasing habitat loss, and sedimentation. However, knowledge on host species is lacking for nearly 40% of mussel species in the Flint River, limiting the efforts of conservation managers. In this study, we assessed the use of host fish by mussels by identifying the species of naturally encysted mussel larvae and transformed juveniles found on wild-caught fishes. Infested fishes were collected in the summers of 2021 and 2022 and held in laboratory conditions. Glochidia and juvenile mussels were collected as they excised from live hosts and were identified by DNA barcoding with the cytochrome oxidase c subunit I locus. Twenty-eight unique mussel–host relationships were identified, 27 of which were considered novel when cross-referenced to the existing mussel–host databases and peer-reviewed literature. Our data build upon knowledge of host use in unionids and further demonstrate the importance of understanding patterns in wild host use.