In the present study, we explored the use of various optical parameters to detect differences in the composition of the dissolved organic matter (DOM) in a set of lakes that are all located on the Canadian Precambrian Shield, but within which Cu and Ni speciation predictions were previously shown to diverge from measured values in some lakes but not in others. Water samples were collected with in situ diffusion samplers in 2007 (N = 18 lakes) and 2008 (N = 8 lakes). Significant differences in DOM quality were identified between the sampling regions (Rouyn-Noranda, Québec and Sudbury, Ontario) and among lakes, based on dissolved organic carbon concentrations ([DOC]), specific UV absorbance (SUVA 254 ), fluorescence indices (FI), and excitation-emission matrix (EEM) fluorescence measurements. Parallel factor analysis (PARAFAC) of the EEM spectra revealed four components, two of which (C3, oxidized quinone fluorophore of allochthonous origin, and C4, tryptophan-like protein fluorescence of autochthonous origin) showed the greatest inter-regional variation. The inter-lake differences in DOM quality were consistent with the regional watershed characteristics as determined from satellite imagery (e.g., watershed-to-lake surface area ratios and relative percentages of surface water, rock outcrops vegetative cover and urban development). Source apportionment plots, built upon PARAFAC components ratios calculated for our lakes, were used to discriminate among DOM sources and to compare them to sources identified in the literature. These results have implications for other areas of research, such as quantifying lake-to-lake variations in the influence of organic matter on the speciation of trace elements in natural aquatic environments.