River water samples were collected at five sites in the state of Colorado, USA, to assess the impact of municipal and industrial discharges on Ag concentrations and speciation in surface waters. Samples were collected and analyzed for total (unfiltered collections), filtered (0.1 and 0.4 μm), particulate (≥0.45 μm), and colloidal Ag (3 kDa-0.1 μm) using ultraclean protocols. A series of laboratory experiments were conducted to assess bias from sample storage, digestion, and preconcentration protocols. In general, upstream unfiltered and particulate Ag concentrations fell within a fairly narrow range, 3.1 to 21 ng/L and 0.2 to 1.7 μg/g, respectively. Downstream unfiltered and particulate Ag concentrations showed a more broad range, 2.8 to 1,110 ng/L and 0.5 to 104 μLg/g, respectively, and reflected attenuated impacts of Ag-laden discharge effluents. However, Ag concentrations in the 0.1-μm filter-passing fraction 0.8 to 1.2 km downstream from major treatment plant effluents were all below the chronic silver criteria. On average, more than 60% of the 0.1-μm filter-passing Ag was associated with colloidal macromolecular organic matter. Silver concentrations in colloids (μg/g) were, on average, the same as those in suspended particulate matter. The percentage abundance of colloidal Ag was similar to that of dissolved organic carbon, suggesting that strong Ag binding ligands exist in both the colloidal and the particle size fractions, as these macromolecular ligands likely play a major role in Ag speciation.