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Macrophyte (aquatic plant) recovery has occurred in rivers worldwide, but assemblage patterns and habitat requirements are generally not well understood. We examined patterns of species composition and macrophyte abundance in the Upper Mississippi River (UMR), spanning 22 years of monitoring and a period of vegetation recovery. Non-metric multidimensional scaling (NMDS) ordination revealed a gradient of macrophyte abundance and diversity for 25 species, which were associated with water velocity, depth, wind fetch, and water clarity. Three macrophyte genera of ecological and restoration interest (<i>Zizania aquatica</i>, <i>Vallisneria americana</i>, and <i>Sagittaria</i> spp.) occupied different ecological niches. Trends of NMDS values showed that <i>Z. aquatica</i> first co-occurred in shallow areas with <i>Sagittaria</i> spp. but then expanded into deeper, lotic habitats where <i>V. americana</i> often resided. Curve Fit regression analysis identified large areas of significant increases in the relative abundance of <i>V. americana</i> and percent cover of <i>Z. aquatica</i> in several reaches of the UMR from 1998–2019. <i>Sagittaria</i> spp. were more spatiotemporally dynamic, which may indicate specific habitat requirements and sensitivity to environmental gradients. Our analyses showed that these three ecologically important genera are spatiotemporally dynamic but have somewhat predictable habitat associations, which can guide macrophyte management and restoration in the UMR and other large, floodplain rivers.