Tropical rivers are an important source of dissolved organic matter (DOM) to coastal oceans. However, temporal and spatial variability of DOM composition and thus its quality in such rivers, on landscape and basin scales, have not been well documented. In this study, we present data on the spatial distribution of DOM quantity and quality based on source, molecular weight, and composition using optical properties including excitation emission matrix fluorescence with parallel factor analysis. We compared such DOM quantity and quality determinations in main river channels and their tributaries for three river systems of the Guayana Shield, Venezuela. Spatial variabilities of DOM parameters were strongly related to differences in the geological settings of the drainage basins and presumably their associated vegetation cover. Linear relationships between quantitative and qualitative DOM parameters were also evident, suggesting that high DOC concentration correlated with chromophoric dissolved organic matter (CDOM) characteristics of higher molecular weight associated with terrestrial sources, while low DOC concentrations correlated with CDOM characteristics of lower molecular weight associated primarily with microbial sources. Such relationships seem to imply that DOM concentrations and their sources/characteristics may be coupled in the studied fluvial systems. In addition, shifts in DOM compositions between terrestrial and microbial signals were observed with changes in water discharge and in watersheds disturbed by gold mining activities. The observed linkages between, and the changes among DOM quantity and quality, suggest that the biogeochemistry of DOM in tropical rivers may be quite sensitive to climatic and land use change.