Anthropogenically degraded benthic-macroinvertebrate communities (benthos) are one of seven beneficial use impairments (BUIs) in the Niagara River Area of Concern (AOC). Over the last 50 years, upgrades to waste-water treatment, industry closures, and sediment remediations reduced contaminant levels throughout the system. Improvements in benthic communities and sediment toxicity, however, were difficult to assess because there are no comparable reference reaches outside the AOC. A multi-phase study was initiated in 2015 to determine if data from inside the AOC could identify reference conditions, if toxicity and benthic-community data from these sites differed from other AOC sites, and if further remediation efforts were warranted in parts of the AOC. Concentrations or quotients of PAHs, PCBs, dioxins and furans, pesticides, and most metals were below their New York Sediment Class A Guidance Values at 10 sites that were subsequently designated as reference sites. Survival and growth data from Chironomus dilutus and Hyalella azteca bioassays indicated that sediments from only a few individual AOC-impact sites were toxic or significantly different from reference sites, and that mean toxicity at pooled AOC-impact and reference sites did not differ significantly. Similarly, New York Biological Assessment Profile scores and chironomid mentum deformity scores at only a few individual sites differed significantly from corresponding indices at reference sites, but neither metric differed significantly in comparisons between pooled AOC-impact and reference sites. Most analyses indicated that benthic communities were unimpaired and that removal criteria for the benthos BUI were largely met in much of the upper Niagara River AOC.