Land-use change, such as agricultural expansion and urbanization, can affect riverine biological diversity and ecosystem functioning. Identifying the major stressors associated with catchment land-use change is a prerequisite for devising successful river conservation and restoration strategies. Here, we analyzed land-use effects on the fatty acid (FA) composition and concentrations in suspended particulate organic matter (SPOM) along a fourth-order tropical river, the Rio das Mortes. Thereby, we aimed at testing the potential of fatty acids in riverine suspended particulate organic matter (SPOM-FAs) as indicators of land-use change in tropical catchments, and at identifying major human impacts on the biochemical composition of SPOM, which represents an important basal energy and organic matter resource for aquatic consumers. River water SPOM and total FA concentrations ranged between 2.8 and 10.2mgdryweight(DW)L−1 and between 130.6 and 268.2μgDWL−1, respectively, in our study. Urbanization was the only land-use category correlating with both FA composition and concentrations, despite its low contribution to whole catchment (1.5–5.6%) and riparian buffer land cover (1.7–6.6%). Higher concentrations of saturated FAs, especially C16:0 and C18:0, which are the main components of domestic sewage, were observed at sampling stations downstream of urban centers, and were highly correlated to urbanization, especially within the 60m riparian buffer zone. Compared to water chemical characteristics (inorganic nutrients, dissolved oxygen, pH, and specific conductance) and river habitat structural integrity, FA variables exhibited a higher variability along the investigated river and were more strongly correlated to urban land use, suggesting that SPOM-FA profiles may be an efficient indicator of urban land-use impacts on larger tropical rivers. High total FA concentrations in the SPOM of urbanized tropical rivers may represent high-energy biochemical subsidies to food webs, potentially leading to changes in functional ecosystem characteristics, such as bacterial and suspension-feeder production.