The backwater lowland habitats of large rivers, like the Mississippi River in North America, present complex often inaccessible environments for traditional capture-based fish biodiversity sampling. Our knowledge of the assemblages of fishes that occupy such habitats are often incomplete, and this can compromise management efforts. We employed environmental DNA (eDNA) metabarcoding methods to sample a Mississippi River bottom wetland system to assess ichthyofaunal diversity, and assemblage structure across habitat types, and we compared our results to capture-based survey records for the same habitats. We collected water samples in spring and fall of 2022 from slough, ditch, shallow lake and bayou habitats that varied in depth, vegetation, seasonal variability, and connectivity to the Mississippi River channel. We detected a diverse array of fish species that included 51 taxa. Nearly all species previously documented in the habitats were detected by eDNA metabarcoding, and we increased the number of documented species by more than a third. Most of the species were ubiquitous across the range of habitats, but there was also substantial assemblage structure, with some species exhibiting clear habitat specificity. Fall sampling was limited to the deeper bayou habitats where seasonal variation between spring and fall was minimal. eDNA metabarcode sampling was demonstrated to be effective at detecting invasive species as well as uncommon species, which included several species of conservation concern.