Status of several large-river darter species is uncertain because of difficulties sampling deep-water habitats. We characterized the darter (Family: Percidae) community in main and side channel macrohabitats of the upper Mississippi River using a small-mesh benthic trawl at sites in five navigation pools and a portion of the lower St. Croix River encompassing nearly 200 river kilometers in 2016 and 2017. We captured six darter species in conjunction with a survey to assess crystal darter (Crystallaria asprella) (state endangered) status and estimated density (n/700 m2) and population size for selected species in navigation pools. No crystal darter were collected in 83 trawl tows, including tows made at historical crystal darter locations. However, a total of 154 western sand darter (Ammocrypta clara) (globally vulnerable) were captured with density estimates ranging among pools from 1.0 to 9.6 in main channel and 1.0 to 5.0 in side channel macrohabitats. The largest population estimate was 33,286 (95% confidence limit: 17,974–52,725) for western sand darter in all side channels in Pool 7. A total of 71 river darter (Percina shumardi) were captured with zero caught in some pools to a maximum mean density of 17.5/700 m2 in Pool 3. Highest population size was 14,829 (8260–25,127) for river darter in the lower St. Croix River. A total of 43 logperch (Percina caprodes) and 143 johnny darter (Etheostoma nigrum) were captured but exhibited clumped spatial distributions that hindered population estimates. Only three mud darter (Etheostoma asprigene) and one slenderhead darter (Percina phoxocephala) were captured in deep-water habitats. Crystal darter absence supports continued state endangered classification. However, our density and population estimates for western sand darter and river darter represent the first such estimates for the upper Mississippi River and perhaps the world and should be used as baselines for future comparisons.