The discharge of terrestrial dissolved organic matter (DOM) by streams is an important cross-system linkage that strongly influences downstream aquatic ecosystems. Isotopic tracers are important tools that can help to unravel the source of DOM from different terrestrial compartments in the landscape. Here we demonstrate the spatial and seasonal variation of delta34S of DOM in 10 boreal streams to test if the tracer could provide new insights into the origin of DOM. We found large spatial and seasonal variations in stream water delta34S-DOM values ranging from -5.2 per thousand to +9.6 per thousand with an average of +4.0 +/- 0.6 (N = 62; average and 95% confidence interval). Large seasonal variations were found in stream water delta34S-DOM values: for example, a shift of more than 10 per thousand during the spring snowmelt in a wetland-dominated stream. Spatial differences were also observed during the winter base flow with higher delta34S-DOM values in the fourth-order Krycklan stream at the outlet of the 68 km2 catchment compared to the small (< 1 km2) headwater streams. Our data clearly show that the delta34S-DOM values have the potential to be used as a tracer to identify and generate new insights about terrestrial DOM sources in the boreal landscape.