Terrestrial and aquatic dissolved organic matter (DOM) was characterized to trace the likely processes of DOM formation and stream export in a permafrost-dominated watershed in central Siberia. Stream samples were collected in spring (May–June 2003) and summer (July–August 2003) at both low flow and stormflow. Dissolved organic matter was analyzed by pyrolysis/gas chromatography/mass spectrometry, and identified pyrolysis products were simultaneously analyzed for compound-specific isotope ratios by isotope ratio mass spectrometry. Pyrograms of terrestrial and stream DOM contained a similar series of pyrolysis products, suggesting a terrestrial origin for DOM in the small stream draining our study catchment. However, despite the overall similarity of chemical composition of stream DOM at different seasons, we also observed distinct differences in isotopic fingerprint between seasons and hydrologic phases (stormflow versus low flow). This variation appears to be due to the changing origin of stream DOM from different soil layers and the catchment sources following permafrost thawing during the frost-free period. In general, chemical and isotopic composition of stream DOM was similar to DOM produced in soils of colder north facing slopes (P < 0.01) with a shallow active layer. South facing slopes with deeper active layers produce little DOM that enters the stream, suggesting that DOM produced in the active layer is retained and stabilized in underlying, unfrozen mineral soils. Climate change that results in additional seasonal thawing of permafrost-dominated landscapes will decrease the amount of DOM exported to riverine systems and change its chemical composition.