Alpine river and lake systems on the Tibetan Plateau are highly sensitive indicators and amplifiers of global climate change and important components of the carbon cycle. Dissolved organic matter (DOM) encompasses organic carbon in aquatic systems, yet knowledge about DOM variation throughout the river???lake aquatic continuum within alpine regions is limited. We used optical spectroscopy, ultrahigh-resolution mass spectrom-etry (Fourier transform ion cyclotron resonance mass spectrom-etry), and stable water isotopic measurements to evaluate linkages between DOM composition and hydrological connection. We investigated glacial influences on DOM composition throughout the watershed of Selin Co, including upstream glacier-fed rivers and downstream-linked lakes. We found that the dissolved organic carbon concentration increased, whereas specific ultraviolet absorbance (SUVA254) decreased along the river???lake continuum. Relative to rivers, the downstream lakes had low relative abundances of polyphenolic and condensed aromatic compounds and humic-like substances but increased relative abundances of aliphatics and protein-like compounds. SUVA254 decreased while protein -like components increased with enriched stable water isotope ??2H-H2O, indicating that DOM aromaticity declined while autochthonous production increased along the flow paths. Glacier meltwater contributed to elevated relative abundances of aliphatic and protein-like compounds in headwater streams, while increased relative abundances of aromatics and humic-like DOM were found in glacier-fed lakes than downstream lakes. We conclude that changes in hydrological conditions, including glacier melt driven by a warming climate, will significantly alter DOM composition and potentially their biogeochemical function in surface waters on the Tibetan Plateau.