The use of zinc (Zn) isotope ratios as a tracer of anthropogenic contamination has been assessed using an extensive collection of river water samples from the Seine River basin (France) collected between 2004 and 2007. The 66Zn/64Zn ratios (expressed as delta66Zn) of dissolved Zn have been measured by MC-ICP-MS after chemical separation of Zn using an improved technique adapted to large volumes of water. Significant isotopic variations (0.07-0.58 per thousand) occur along a transect from pristine areas of the Seine basin to the estuary and with time in Paris, and are coherent with the Zn enrichment factor. Dissolved Zn in the Seine River displays conservative behavior, making Zn isotopes a good tracer of the different sources of contamination. Dissolved Zn in the Seine River is essentially of anthropogenic origin (> 90%) compared to natural sources (< 7%). Roof leaching from Paris conurbation is a major source of Zn, characterized by low delta66Zn values that are distinct from other natural and anthropogenic sources of Zn. Our study highlights the absence of distinctive delta66Zn signatures of fertilizer, compost or rain in river waters of rural areas, and therefore suggests the strong retention of Zn in the soils of the Basin. Our study demonstrates that Zn isotope ratios will be a powerful new tool to trace pathways of anthropogenic Zn in the environment.