The EPA's Environmental Monitoring and Assessment Program large-river assessment protocol was applied to assess the ecological condition, major stressors, and likely human disturbances of the mainstem Malheur River, OR. We used inflatable rafts to allow launching and retrieving from difficult access points and to sample river reaches inaccessible to most other boat types or wading crews, including areas with river obstacles such as rapids and small dams. Electrofishing twenty-four 1–2 km long reaches within the lower 150 km of the river during the summers of 2006 and 2007 revealed: (1) the absence of native mountain whitefish Prosopium williamsoni; (2) the presence of previously undocumented endemics, mountain sucker Catostomus platyrhynchus and leopard dace Rhinichthys falcatus; (3) the existence of previously undocumented aliens, flathead catfish Pylodictis olivaris, tadpole madtom Noturus gyrinus, pumpkinseed Lepomis gibbosus, fathead minnow Pimephales promelas, and western mosquitofish Gambusia affinis; (4) possible range extensions into the main river by two alien basin-reservoir inhabitants, largemouth bass Micropterus salmoides and yellow perch Perca flavescens; and (5) index of biological integrity scores that declined from a high of 53 for an upstream site to 0.5 for a site 6 km from the river mouth. Regular standardized direct assessments of large-river fish assemblages can provide important information used to update river-basin management plans and inform water-resource managers.