Our study develops and demonstrates an objective method for selecting reference sites for the assessment of ecological condition in freshwater ecosystems. The method uses widely available GIS data to group potential sites based on their natural environments. It then establishes the degree and types of human activities each site is exposed to prior to scoring the sites in each group by the relative amount of human activity present. Finally, the sites in each group with the least amount of human activity are categorized as reference sites, with the boundary between reference and test sites defined to maximize the distinctiveness of the two categories with respect to human activity. Application of this technique for the purpose of identifying headwater reference basins in rural areas of southwestern Ontario resulted in the classification of basins into six natural groups based on the dominant texture of the surface geology. Development of a human activity gradient indicated that basins varied according to the amount of exposure to agricultural activities with most basins having at least moderate exposure. Establishment of the reference test boundary indicated that the selected reference basins exhibited substantively lower extents of agricultural activity than test sites for most groups. Because this method uses only widely available GIS data, it enables rapid and cost-effective identification of candidate reference sites, even for large, remote, and understudied regions.