Conservation of lotic fishes requires consideration of complex patterns among a changing mosaic of fish assemblage structure at multiple spatial scales. Studies have demonstrated the influence of localized competition and predation among stream fishes, but researchers have been largely unable to determine to what extent interspecific interactions act to control fish assemblage structure. Recent declines to geographic range and abundance of Plains Topminnow (Fundulus sciadicus (Cope)) have been anecdotally attributed to altered fish assemblages and potential competitive or predatory associations; however, important species interactions have yet to be identified. Variation among fish assemblage structure at 473 locations known to historically support Plains Topminnow was described in Nebraska to demonstrate the role of assemblage dynamics in regulating species occurrence. Nonrandom patterns of fish assemblage composition in terms of Plains Topminnow persistence were apparent statewide, and multiple relationships were recognized as regionally influential (e.g., Niobrara River: Largemouth Bass Micropterus salmoides (Lacépède)). Strong correlations were described relating Plains Topminnow extirpation to nonnative species establishment (Common Carp Cyprinus carpio (Linnaeus), Western Mosquitofish Gambusia affinis (Baird and Girard)), and several potential predatory (Esocidae, Channel Catfish Ictalurus punctatus (Rafinesque)) or competitive interactions (Western Mosquitofish, Bluegill Lepomis macrochirus (Rafinesque), Sand Shiner Notropis stramineus (Cope), Red Shiner Cyprinella lutrensis (Baird and Girard)) were identified. Positive relationships are attributed to similar reliance of particular abiotic conditions (Emerald Shiner Notropis atherinoides (Rafinesque)) or habitat types (Gasterosteidae, Green Sunfish Lepomis cyanellus (Rafinesque), Iowa Darter Etheostoma exile (Winn)). Other identified species will require research to elucidate potential biotic interactions with Plains Topminnow. Resultant models are hypothesized to reflect biotic processes, and thereby provide analytical insight into the long-term influence of fish assemblage structure on a rare species. Identified relationships provide preliminary recognition of potentially important interspecific interactions involving an unfamiliar species from which further hypotheses can be evaluated to ascertain casual relationships.