Climate warming and permafrost degradation at high latitudes will likely impact watershed hydrology, and consequently, alter the concentration and character of dissolved organic carbon (DOC) in northern rivers. We examined seasonal variation of DOC chemistry in 16 streams of the Yukon River basin, Alaska. Our primary objective was to evaluate the relationship between source water (shallow versus deep groundwater flow paths) and DOC chemical composition. Using base cation chemistry and principal component analysis, we observed high contributions of deep groundwater to glacial and clearwater streams, whereas blackwater streams received larger contributions from shallow groundwater sources. DOC concentration and specific ultraviolet absorbance peaked during spring snowmelt in all streams, and were consistently higher in blackwater streams than in glacial and clearwater streams. The hydrophobic acid fraction of DOC dominated across all streams and seasons, comprising between 35% and 56% of total DOC. The hydrophilic acid fraction of DOC was more prominent in glacial (23% ± 3%) and clearwater streams (19% ± 1%) than in blackwater streams (16% ± 1%), and was enriched during winter base flow (29% ± 1%) relative to snowmelt and summer base flow. We observed that an increase in the contribution of deep groundwater to streamflow resulted in decreased DOC concentration, aromaticity, and DOC-to-dissolved organic nitrogen ratio, and an increase in the proportion of hydrophilic acids relative to hydrophobic acids. Our findings suggest that future permafrost degradation and higher contributions of groundwater to streamflow may result in a higher fraction of labile DOM in streams of the Yukon basin.