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Abstract Environmental DNA (eDNA), i.e. DNA released in the environment by any living form, represents a formidable opportunity to gather high-throughput and standard information on the distribution or feeding habits of species. It has therefore great potential for applications in ecology and biodiversity management. However, this research field is fast-moving, involves different areas of expertise and currently lacks standard approaches, which calls for an up-to-date and comprehensive synthesis. Environmental DNA for biodiversity research and monitoring covers current methods based on eDNA, with a particular focus on “eDNA metabarcoding”. Intended for scientists and managers, it provides the background information to allow the design of sound experiments. It revisits all steps necessary to produce high-quality metabarcoding data such as sampling, metabarcode design, optimization of PCR and sequencing protocols, as well as analysis of large sequencing datasets. All these different steps are presented by discussing the potential and current challenges of eDNA-based approaches to infer parameters on biodiversity or ecological processes. The last chapters of this book review how DNA metabarcoding has been used so far to unravel novel patterns of diversity in space and time, to detect particular species, and to answer new ecological questions in various ecosystems and for various organisms. Environmental DNA for biodiversity research and monitoring constitutes an essential reading for all graduate students, researchers and practitioners who do not have a strong background in molecular genetics and who are willing to use eDNA approaches in ecology and biomonitoring.