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Aquatic ecosystems are particularly vulnerable to the introduction of non-indigenous species (NIS), leading to multi-faceted ecological, economic and health impacts worldwide. The Iberian Peninsula comprises an exceptionally biodiverse Mediterranean region with a high number of threatened and endemic aquatic species, most of them strongly impacted by biological invasions. Following a structured approach that combines a systematic review of available information and expert opinion, we provide a comprehensive and updated multi-taxa inventory of aquatic NIS (fungi, macroalgae, vascular plants, invertebrates and vertebrates) in Iberian inland waters. Moreover, we assess overall patterns in the establishment status, introduction pathways, native range and temporal introduction trends of listed NIS. In addition, we discuss the legal coverage provided by both national (Spanish and Portuguese) and European NIS regulations. We inventoried 326 aquatic NIS in Iberian inland waters, including 215 established, 96 with uncertain establishment status and 15 cryptogenic taxa. Invertebrates (54.6%) and vertebrates (24.5%) were the groups with the highest number of NIS, with Arthropoda, Mollusca, and Chordata being the most represented phyla. Recorded NIS originated from diverse geographic regions, with North and South America being the most frequent. Vertebrates and vascular plants were mostly introduced through intentional pathways (i.e. release and escape), whereas invertebrates and macroalgae arrived mostly through unintentional ways (i.e. contaminant or stowaway). Most of the recorded NIS were introduced in Iberian inland waters over the second half of the 20th century, with a high number of NIS introductions being reported in the 2000s. While only 8% of the recorded NIS appear in the European Union list of Invasive Alien Species of Union concern, around 25% are listed in the Spanish and Portuguese NIS regulations. This study provides the most updated checklist of Iberian aquatic NIS, meeting the requirements set by the EU regulation and providing a baseline for the evaluation of its application. We point out the need for coordinated transnational strategies to properly tackle aquatic invasions across borders of the EU members. [ABSTRACT FROM AUTHOR]