Human activities introduce a variety of chemicals to the Laurentian Great Lakes including pesticides, pharmaceuticals, flame retardants, plasticizers, and solvents (collectively referred to as contaminants of emerging concern or CECs) potentially threatening the vitality of these valuable ecosystems. We conducted a basin-wide study to identify the presence of CECs and other chemicals of interest in 12 U.S. tributaries to the Laurentian Great Lakes during 2013 and 2014. A total of 292 surface-water and 80 sediment samples were collected and analyzed for approximately 200 chemicals. A total of 32 and 28 chemicals were detected in at least 30% of water and sediment samples, respectively. Concentrations ranged from 0.0284 (indole) to 72.2 (cholesterol) μg/L in water and 1.75 (diphenhydramine) to 20,800 μg/kg (fluoranthene) in sediment. Cluster analyses revealed chemicals that frequently co-occurred such as pharmaceuticals and flame retardants at sites receiving similar inputs such as wastewater treatment plant effluent. Comparison of environmental concentrations to water and sediment-quality benchmarks revealed that polycyclic aromatic hydrocarbon concentrations often exceeded benchmarks in both water and sediment. Additionally, bis(2-ethylhexyl) phthalate and dichlorvos concentrations exceeded water-quality benchmarks in several rivers. Results from this study can be used to understand organism exposure, prioritize river basins for future management efforts, and guide detailed assessments of factors influencing transport and fate of CECs in the Great Lakes Basin.