American eel populations are declining and have recently become a species of interest by the United States Fish and Wildlife Service to list as a threatened species. However, the American eel population in the largest inland lotic waterway in North America (Mississippi River) has received little attention despite the apparent relevance. Because of the lack of information on the Mississippi, we evaluated trends in relative abundance and habitats occupied by American eel using long-term data collected on the Mississippi River (i.e., Upper Mississippi River Restoration – Environmental Management Program). During the 18 y study, a total of 92 American eels were collected throughout the Upper Mississippi River (Lake City, Minnesota downstream to Cape Girardeau, Missouri) with a relatively fewer individuals captured as of recent. Across macrohabitats, unstructured and structured (i.e., diked) main channel borders had the greatest number of American eels captured; however, eels were infrequently captured in impounded habitats. In terms of mesohabitat use, most American eels were captured in areas characterized by the shallowest waters, rock substrates, and low velocities. We believe the information provided in this study will promote American eel conservation in the Mississippi River.