Freshwater mussels are important components of aquatic systems, where they cycle nutrients, stabilize streambeds, increase niche habitats, and are sources of food for mammals, birds, fish, and aquatic invertebrates. The biogeographically distinct Ozark Highlands is one of the oldest terrestrial regions on the planet and is home to over 200 endemic species including freshwater mussels. Key to the conservation and management of this mussel fauna is documenting their distribution and determining if any changes have occurred. In this study, our objectives were to document the diversity and distribution of the mussel fauna of the Missouri portion of the Eleven Point River, a National Wild and Scenic River, and determine if any changes had occurred over a 25+ yr period. During 1982–1985 and 2012–2019 we surveyed 29 locations, including 12 surveyed during both periods, in summer to early fall, during low flow conditions with timed visual and tactile searches. We calculated metrics to measure the status, composition, and abundance of the mussel community, and examined the rate populations were experiencing extirpation or colonization. Historically, 21 freshwater mussel species were known from Eleven Point River; 20 were observed live or as shell material in the present surveys. During both surveys, the Eleven Point River basin was dominated by members of the Lampsilini, which exhibit periodic life history strategies. We detected significant declines in the number of live individuals, CPUE, and richness at the 12 resurveyed locations. Freshwater mussel species in the Eleven Point River are being extirpated at a significantly higher rate than colonizing areas of the Eleven Point River. Causes for this decline remain unknown.