<p>Dissolved organic matter (DOM) in large rivers is a key component of the global carbon cycle. Spatial distribution of molecular signatures of DOM over the upper reach of the Changjiang River has not been well examined. A total of six surface water DOM samples were collected from Tuotuo River to the Three Gorges Dam (~ 3500&#xa0;km long) along the upper reach of the Changjiang River during the flood season in 2019, and were characterized by multi-methods. Fluorescence spectroscopy analysis revealed that all the DOM was mainly terrestrial sourced. Lignin phenol marker analysis revealed that the lignin in the DOM from Tuotuo River and Zhimenda, the two sites in the headwater region on high-altitude grassland, was dominated by non-woody angiosperm tissues. Alternatively, the lignin in the DOM from the Three Gorges Dam had the highest organic carbon (OC)-normalized lignin content. FT-ICR MS analysis showed that the DOM in Tuotuo River had the highest relative abundances of N/S-containing molecules as well as biolabile aliphatic and carbohydrate-like molecules resulting from the intense meltwater erosion. The photolabile highly aromatic and polycyclic aromatic molecules gradually accumulated in the upstream and peaked in Sanduizi due to wildfire inputs, followed by decrease at the downstream sites stemming from enhanced photodegradation. The relative abundance of lignin-like molecules kept increasing along the river due to their recalcitrant property and the continuous input from forest and cropland vegetation. The results demonstrated the complex spatial distribution and key influencing factors of molecular signatures of surface water DOM along the upper reach of the Changjiang River.</p> Graphical Abstract <p></p>