Urban stormwater is a conduit for pollutants such as nutrients and tire-wear compounds through streams to the Great Lakes receiving waters. The transformation product of tire antiozonant 6PPD, 6PPD-quinone (6PPD-Q), is implicated in causing fish mortality. Urban streams entering Lake Ontario and waters adjacent to urban centers in Toronto and Hamilton, Ontario, were assessed for 6PPD-Q and its potential to impact aquatic life. Sampling occurred during fall and spring dry-and wet-weather flows in 2021−2022, overlapping with fish spawning times. Concentrations of 6PPD-Q were elevated during wet-weather (maximum 82 ng/ L) in streams with the greatest degree of urbanization and road density but were much lower (2.4−4.5 ng/L) in nearshore waters, suggesting rapid dilution. Precipitation, road density within watersheds, chloride (from road salts), and total phosphorus were correlated with 6PPD-Q concentrations, suggesting that such parameters may provide a means to identify and prioritize key stream habitats for further assessment. A screening level hazard assessment using application factors of 10−1000 and available toxicity end points for fish (rainbow trout, brook trout, coho salmon) showed that 6PPD-Q concentrations may pose a risk in urban streams and nearshore waters of the region. We recommend that stormwater quality control measures be assessed for their effectiveness in reducing 6PPD-Q concentrations.