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© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Kirkels, F. M. S. A., Ponton, C., Galy, V., West, A. J., Feakins, S. J., & Peterse, F. From Andes to Amazon: assessing branched tetraether lipids as tracers for soil organic carbon in the Madre de Dios River system. Journal of Geophysical Research-Biogeosciences, 125(1), (2020): e2019JG005270, doi:10.1029/2019JG005270., We investigate the implications of upstream processes and hydrological seasonality on the transfer of soil organic carbon (OC) from the Andes mountains to the Amazon lowlands by the Madre de Dios River (Peru), using branched glycerol dialkyl glycerol tetraether (brGDGT) lipids. The brGDGT signal in Andean soils (0.5 to 3.5 km elevation) reflects air temperature, with a lapse rate of −6.0 °C/km elevation (r 2 = 0.89, p < 0.001) and −5.6 °C/km elevation (r 2 = 0.89, p < 0.001) for organic and mineral horizons, respectively. The same compounds are present in river suspended particulate matter (SPM) with a lapse rate of −4.1 °C/km elevation (r 2 = 0.82, p < 0.001) during the wet season, where the offset in intercept between the temperature lapse rates for soils and SPM indicates upstream sourcing of brGDGTs. The lapse rate for SPM appears insensitive to an increasing relative contribution of 6‐methyl isomer brGDGTs produced within the river. River depth profiles show that brGDGTs are well mixed in the river and are not affected by hydrodynamic sorting. The brGDGTs accumulate relative to OC downstream, likely due to the transition of particulate OC to the dissolved phase and input of weathered soils toward the lowlands. The temperature‐altitude correlation of brGDGTs in Madre de Dios SPM contrasts with the Lower Amazon River, where the initial soil signature is altered by changes in seasonal in‐river production and variable provenance of brGDGTs. Our study indicates that brGDGTs in the Madre de Dios River system are initially soil derived and highlights their use to study OC sourcing in mountainous river systems., The brGDGT analyses were supported by NWO‐Veni grant 863.13.016 to F.P. This material is based upon work supported by the US National Science Foundation under grant EAR‐1227192 to A. J. W. and S. J. F. for the river fieldwork and lipid purification. In Perú, we thank the Servicio Nacional de Áreas Naturales Protegidas por el Estado (SERNANP) and personnel of Manu and Tambopata National Parks for logistical assistance and permission to work in the protected areas. We thank the Explorers' Inn and the Pontifical Catholic University of Perú (PUCP), as well as the Amazon Conservation Association for the use of the Tambopata and Wayqecha Research Stations, respectively. For river fieldwork assistance, we thank M. Torres, A. Robles, and A. Cachuana. Soil samples were contributed by Andrew Nottingham and Patrick Meir. Logistical support was provided by Y. Malhi, J. Huaman, W. Huaraca Huasco, and other collaborators as part of the Andes Biodiversity and Ecosystems Research Group ABERG (www.andesresearch.org). We thank Dominika Kasjaniuk for technical support at Utrecht. Two anonymous reviewers have provided valuable comments that have helped to improve this manuscript. Geochemical and brGDGT data are available in the PANGAEA Data Repository (Kirkels et al., 2019) and can be accessed at https://doi.pangaea.de/10.1594/PANGAEA.906170