Colloidal organic matter (COM) collected in small and large watercourse tributaries of the Negro River (Brazil) were fractionated and characterized by an asymmetrical flow field-flow fractionation (AFlFFF) coupled to UV and seven angle laser light scattering (LS) detectors, and by size exclusion chromatography (SEC) coupled with a UV detection. Number and weight average molar masses, weight average gyration radiuses RGw and polydispersity indexes were obtained for each sample in two separate runs under conditions optimized for lower and larger size fractions. The results demonstrate the existence of a decrease of size of the colloidal matter when passing from first order streams to higher order rivers. No significant changes were found in size distributions of samples collected during the low and high flow stages at the same site. The influence of selected pre-treatments such as filtration and reverse osmosis pre-concentration on the size and molar mass distributions was also studied.