AbstractClimate change is decreasing watershed glacial coverage throughout Alaska, impacting the biogeochemistry of downstream ecosystems. We collected streamwater fortnightly over the glacial runoff period from three streams of varying watershed glacier coverage (0–49%) and a subglacial outflow to assess how glacier recession impacts the relative contributions of glacier and terrestrial plant derived dissolved organic matter (DOM) inputs to streams. We show an increase in the fraction of old dissolved organic carbon (up to ∼ 3200 yr old radiocarbon age) with increasing glacial meltwater contribution to streamflow. We use a dual isotopic mixing model (δ13C and Δ14C) to quantify the relative contribution of terrestrial and glacial sources to streamwater DOM. The endmember contributions were further compared to DOM molecular compositional data from Fourier‐transform ion cyclotron resonance mass spectrometry to assess whether DOM composition can be linked to streamwater DOM source in watersheds with varying contributions of glacial runoff. This approach revealed the glacial fraction was positively correlated with percent relative abundance of heteroatom‐containing DOM molecular formulae, aliphatics, and peptide‐like formulae, while the terrestrial fraction was positively correlated with condensed aromatics and polyphenolics. These results provide information about how the retreat of mountain glaciers will impact the composition and thus biogeochemical role of DOM delivered to downstream ecosystems. Our findings highlight that combining an isotopic mixing model and ultrahigh resolution mass spectrometry data can provide novel insights into how changes in watershed landcover impact the source and chemical properties of streamwater DOM.