Waste materials from mining of the Cerro Rico de Potosí precious metal-polymetallic tin deposits of southern Bolivia have been released to the headwaters of the Rio Pilcomayo for the past 450 years, resulting in extensive contamination of water, sediments and soils along the upper reaches of the river. This study uses isotopic data to identify the primary sources of Pb to the aquatic environment, and the relative contributions of each source to pre and post-mining alluvial deposits. Prior to the onset of mining activities in 1545, alluvial sediments along the Rio Pilcomayo were dominated by Pb from the underlying bedrock and from mineralized rocks exposed at the surface of Cerro Rico. Mining and milling operations at Cerro Rico released a new source of Pb to the river that can be traced downstream for at least 200 km. Simple mixing models suggest that Pb from the mines comprise between 30 and 89% of the Pb in the modern channel bed sediment. However, these estimates may be low because the isotopic composition of the contaminant source was based on samples of ore deposits rather than mill tailings, the latter of which contain fragments of both ore and host rock.