The objectives of the Environmental Monitoring and Assessment Program for Great River Ecosystems (EMAP-GRE) are to (1) develop and demonstrate, in collaboration with states, an assessment program yielding spatially unbiased estimates of the condition of mid-continent great rivers; (2) evaluate environmental indicators for assessing great rivers; and (3) assess the current condition of selected great river resources. The purpose of this paper is to describe EMAP-GRE using examples based on data collected in 2004–006 with emphasis on an approach to determining reference conditions. EMAP-GRE includes the Upper Mississippi River, the Missouri River, and the Ohio River. Indicators include biotic assemblages (fish, macroinvertebrates, plankton, algae), water chemistry, and aquatic and riparian physical habitat. Reference strata (river reaches for which a single reference expectation is appropriate) were determined by ordination of the fish assemblage and examination of spatial variation in environmental variables. Least disturbed condition of fish assemblages for reference strata was determined by empirical modeling in which we related fish assemblage metrics to a multimetric stressor gradient. We inferred least disturbed condition from the y-intercept, the predicted condition when stress was least. Thresholds for dividing the resource into management-relevant condition classes for biotic indicators were derived using predicted least disturbed condition to set the upper bound on the least disturbed condition class. Also discussed are the outputs of EMAP-GRE, including the assessment document, multimetric indices of condition, and unbiased data supporting state and tribal Clean Water Act reporting, adaptive management, and river restoration.