1. To evaluate patterns in mussel assemblages in the Upper Mississippi River (UMR), data from systematic surveys of mussels conducted in three large reaches (Navigation Pools 5, 6, and 18) from 2005–2007 were analysed. 2. Nonmetric multi-dimensional scaling analyses and permutation tests indicated that assemblages differed among reaches. The mussel assemblage in Pool 18 was substantially different from the assemblage in Pool 5 and moderately different from the assemblage in Pool 6, whereas assemblages in Pools 5 and 6 were similar. Assemblages in broadly defined, flowing aquatic habitats did not substantially differ. 3. The dissimilarity of Pool 18 was primarily the result of Pool 18 having higher abundances of three Quadrula species (Q. quadrula, Q. pustulosa, and Q. nodulata), and lower abundances of Amblema plicata and Fusconaia flava. 4. Rarefaction analyses showed that species richness and species density were higher in Pool 18 compared with the other two pools. 5. Large-scale patterns in mussel assemblages may be related to other longitudinal trends in the system including geomorphology, water quality, and abundances of fish species that serve as hosts for glochidial larvae. 6. The results suggest that management goals and actions in the UMR may need to account for important differences in mussel assemblages that occur among reaches.