Spatial hierarchical approaches to classify freshwater systems can add to our understanding of biogeographical patterns and can be used for biodiversity conservation planning. The Strawberry River is located primarily in the Ozark Highlands Central Plateau of north central Arkansas, USA, with a small downstream portion in the Mississippi Alluvial Plain and has been designated an Extraordinary Resource Water, an Ecologically Sensitive Water Body, and a Natural Scenic Waterway. The goals of this study were to document Strawberry River, Arkansas freshwater mussels to aid in conservation planning. Our first objective was to inventory freshwater mussel species in the Strawberry River. Our second objective was to use this stream-wide dataset to classify the freshwater mussel assemblages. We used unpublished survey data of 59 sites distributed from the headwaters to the mouth to inventory species occurrence and abundance, classified mussel assemblages using non-metric multi-dimensional scaling (NMS), and conducted indicator species analysis on resulting assemblages. We observed 39 taxa across the 59 survey sites including two S1, five S2, 16 S3, 11 S4, four S5, and one state non-ranked conservation rank species. Furthermore, our assemblage NMS revealed two distinct freshwater mussel assemblages roughly organized by an upstream (Sites 1–31) to downstream (Sites 32–59) gradient. There were five upstream indicator species and 13 downstream indicator species. This study provides a case study on using existing datasets with NMS and indicator species analyses to classify mussel assemblages and adds to our understanding of freshwater mussel fauna classification at smaller spatial scales. Both NMS and indicator species outcomes can aid in conservation planning for freshwater mussels.