Surface sediments from a 100-km stretch of the River Clyde, UK, and its estuary were analysed for glycerol dialkyl glycerol tetraethers (GDGTs) to track the downstream changes in the source of organic matter (OM) and to evaluate the impact of urbanisation. Bacterial membrane lipids, named branched GDGTs (brGDGTs), produced in soils and rivers ranged from 1.6 to 58μgg−1 organic carbon (OC) and the isoprenoid GDGT crenarchaeol, mainly from marine archaea, ranged from 0.01 to 42μgg−1 OC. The highest brGDGT concentrations were in the upper river, in Glasgow city and in the outer estuary, suggesting higher soil-derived OM input. By contrast, crenarchaeol concentrations gradually increased from the tidal weir in Glasgow towards the sea. This spatial distribution of the tetraethers was reflected in the branched and isoprenoid tetraether (BIT) index, a proxy for soil versus marine carbon. The highest BIT values (1.0) occurred upstream, estuarine values ranged from 0.9 to 0.6 and the lowest values (0.4) were found in the outer estuary. An independent proxy for soil-derived OM, stable carbon isotope (δ13C) values, showed a comparable decrease in terrigenous OM contribution towards the sea, but was more variable compared to the BIT. Conversely, carbon/nitrogen (C/N) showed a constant trend, suggesting that it is not a reliable indicator of OM source in the Clyde. Neither BIT, δ13C nor C/N were able to reflect the input of urban effluents from Glasgow.