Organic matter (OM) plays a major role in driving both abiotic and biotic processes within aquatic ecosystems, and these in turn depend on the quantity and characteristics of the OM present. This study aimed to determine temporal and spatial variability of organic matter within the River Derwent catchment by conducting monthly sampling over 2 years, at 14 sites along the River Derwent and its tributaries, Tasmania, Australia. Dissolved organic matter (DOM) was characterised using absorbance and fluorescence spectroscopy. TON and TOC concentrations varied temporally and spatially and ranged from 0.05 – 1.2 mg/L and 1.2 - 19 mg/L, respectively. Fluorescent Excitation Emission Scans followed by PARAFAC analysis revealed presence of four DOM components: two Fulvic-like, one Humic-like and one Protein-like. DOM composition and characteristics were shown to differ temporally, spatially and seasonally and were directly related to flow and land use. Fish farms also altered the organic nitrogen concentrations and the C/N ratio. Results from this study provide insight into factors controlling OM dynamics within the River Derwent catchment and suggest the potential use of organic nitrogen as an indicator of aquaculture effects in freshwaters.