The plains of midwestern North America have undergone significant anthropogenic alterations following European settlement with consequent effects to lotic fish assemblage structure. We examined trends in fish assemblage structure and function in Nebraska's lotic systems using site-specific, presence-absence data from historical (1939–1940) and contemporary surveys (2003–2005; n  =  183). Shifts in fish assemblage structure were characterized by declines of specialist species (e.g., western silvery minnow Hybognathus argyritis) and increases in nonnative, sport, and generalist species (e.g., common carp Cyprinus carpio). Our research illustrates differences between historical and contemporary surveys for both taxonomic and functional metrics. Changes in fish assemblage structure were correlated with a contemporary measure of anthropogenic alteration (Human Threat Index; HTI) and were most pronounced for large-scale threats (i.e., watershed HTI, overall HTI). The HTI is a composite index of cumulative anthropogenic alterations experienced by a stream system and was used to investigate broad-scale implications of anthropogenic activity on fish assemblage structure. Fish assemblages among sites were more similar in contemporary surveys than in historical surveys, such changes might indicate a homogenization of the fish assemblages. Losses of native species and increases in introduced species have occurred in Nebraska's lotic systems across a broad temporal span and shifts are likely related to high levels of human perturbation.