Background: The change in the optical parameters of dissolved organic matter (DOM) from natural water samples of Pirita River in Estonia were studied during 2 years of winter-spring season. At present, the relative quantitative and qualitative composition of DOM components leached from soils to water, in relation to air and water temperatures and water level fluctuations, has not been estimated in Estonia. The aim was to characterise and fractionate DOM using high-performance size-exclusion chromatography with multiple wavelength detection (DAD) and ultraviolet-visible (UV-vis) spectroscopy. Changes in several chromatographic/molecular and spectroscopic/optical parameters were investigated. Results: The experimental data indicated that the optical and molecular properties of DOM depend on the climate conditions, but the effect can be diverse. In cold climates, the concentration of DOM in river water can be lower (2013) or higher (2014) than in warmer climate conditions. Optical properties indicate that the quantity of humic substances (HS) is minimal under an ice cover and DOM contains more microbial-derived peptide-like constituents. Conclusions:The results point out the usefulness of HPLC-SEC and UV-vis spectroscopy for climate change-related DOM studies in real environmental conditions. Used methods enable detailed monitoring of humic molecules dissolved from the catchment soil, as well as microbial constituents (peptide-like components of DOM). The used approach can be additionally applied for monitoring the changes of raw water quality and thus for the design and optimisation of drinking water process technology.