AbstractDissolved organic matter (DOM) is a key variable influencing aquatic ecosystem processes. The concentration and composition of DOM in streams depend on both the delivery of DOM from terrestrial sources and on aquatic DOM production and degradation. However, there is limited understanding of the variability of stream DOM composition at continental scales and the influence of landscape characteristics and disturbances on DOM across different regions. We assessed DOM composition in 52 streams at seven research sites across six forested ecozones in Canada in 2019–2022 using 26 indices derived from five analytical approaches: absorbance and fluorescence spectroscopy, liquid chromatography—organic carbon detection, Fourier‐transform ion cyclotron resonance mass spectrometry, and asymmetric flow field‐flow fractionation. Combined analyses showed clear clustering and redundancy across analytical techniques, and indicated that compositional variations were primarily related to three axes of DOM composition: (a) aromaticity, which was greater in low‐relief, wetland‐dominated catchments, (b) oxygenation, which was greater in colder and drier ecozones, and (c) biopolymer content, which was greater in lake‐influenced catchments. Variability in DOM composition among research sites was greater than variability of streams within a site and variability over time within a stream. Forest harvesting and wildfire disturbances had no common influence on DOM composition across research sites, emphasizing the need for regional studies. Our study provides a broad understanding of the variability of stream DOM composition and its associations with landscape and catchment characteristics at a subcontinental scale, and provides key insights for the choice and interpretation of DOM indices from various analytical approaches.