Streams in the northeastern US receive mercury (Hg) from direct and indirect atmospheric deposition, as well as current and (or) historic point source contamination / discharges, which, together, contribute to high concentrations in fish throughout the region. Mercury stable isotope ratios were used to identify Hg sources to fish and bed sediments in 23 small streams spanning a wide range in water-quality characteristics and land use, ranging from largely undeveloped, forest-dominated to dense urban with current or historical industrial land use. Median mass-dependent isotopes (δ²⁰²Hg) in prey and game fish differed among three land-use defined groups of sites. Median δ²⁰²Hg values were relatively depleted (-0.95 and -0.83 ‰, respectively) in the forested group, intermediate (-0.61 and -0.58‰, respectively) in the residential group, and relatively enriched (-0.37 and -0.25 ‰, respectively) in the industrial group. A principal components analysis of environmental variables produced a synthetic urban-intensity gradient of increasing chloride, nitrogen, phosphorous, and total mercury concentrations in stream water, total mercury concentrations in bed sediments, and watershed road density. Prey fish and game fish δ²⁰²Hg were strongly correlated to site scores along this urban-intensity gradient (ρ= 0.87, p<0.0001 and ρ= 0.79, p < 0.0001, respectively), indicating that Hg source signatures from the landscape are preserved in fish. In contrast, neither prey fish nor game fish Δ¹⁹⁹Hg (an indicator of photochemistry) were significantly correlated with the urban-intensity gradient, but Δ¹⁹⁹Hg in bed sediment was strongly correlated (ρ= 0.89, p < 0.0001) with this environmental gradient. This indicates that photochemical tracers in sediment are unique to the source of Hg whereas those in fish record more recent water column processes, such as water column demethylation. Using measurements of physical and chemical parameters of stream ecosystems coupled to Hg isotope ratio measurements allows for the identification of Hg source portfolios in fish tissue that can be used to probe Hg bioaccumulation over a wide range of ecosystems.