Abundance of fishes of the main channels of the upper Mississippi River system and of other large North American rivers is largely unknown because historic sampling methods have been inadequate. We used a bottom trawl to estimate spatial and temporal patterns in abundance in the navigation channels of Pool 26 of the Mississippi River and the lower Illinois River. Total biomass density averaged 21 and 29 kg·ha-1 in the navigation channels of Pool 26 and the lower Illinois River, respectively. We identified spatial and temporal patterns in catches of key species using a generalized linear model based on the negative binomial distribution. Some species, including shovelnose sturgeon (Scaphirhynchus platorynchus), are persistent residents of the main channel. Multiple-season residents, including freshwater drum (Aplodinotus grunniens), rely heavily on the main channel during most of the year but leave it briefly, for example to seek thermal refugia in backwaters during winters. We suggest revision of the prevailing notion that main channels of large temperate rivers serve mainly as corridors for movement among other habitat types.