Monitoring plans using environmental DNA have the potential to offer a standardized and cost-efficient method to survey biodiversity in aquatic ecosystems. Among these ecosystems, coastal wetlands are key elements that serve as transition zones between marine and freshwater ecosystems and are today the target of many conservation and restoration efforts. In this sense, eDNA monitoring could provide a rapid and efficient tool for studying and generating baseline biodiversity information to guide coastal wetland management programs. Here, we test an eDNA metabarcoding assay as a tool to characterize vertebrate biodiversity in one of the largest coastal wetlands of Chile, the Rio Cruces Wetland, a Ramsar designated site since 1981. We sampled surface water from 49 sites along the entire wetland. Our eDNA approach detected 91genera of vertebrates including amphibians, fishes, mammals, and birds, as well as identified several cryptic, exotic, and endangered species. Our results also indicated that the spatial distribution of eDNA from different species is spatially structured despite the complex hydrodynamics inherent in this wetland due to the influence of daily tidal regimes. For amphibians and fishes, the number of taxa detected with eDNA was higher in the periphery of the wetland, and increased with proximity to the ocean, a pattern consistent with small-scale spatial sensitivity for some species and eDNA accumulation downstream for others. Birds and mammals showed somewhat more idiosyncratic distributions. Taken together, our results add to the growing body of evidence showing eDNA can serve as a rapid cost-effective tool