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Freshwater mussels of the order Unionida have been dramatically declining globally. Despite their ecological importance, conservation of these animals has been hindered by unresolved taxonomy and a lack of data on the distribution and status of populations, especially in southern Europe. Although the Italian Peninsula has been noted as a centre of endemism and one of the major refugia of the glacial ages for several taxa, few studies have been performed on the genetic diversity of Unionida. Most importantly, the taxonomic status of several freshwater mussel populations of the Italian Peninsula is still unresolved. Here we present the first comprehensive dataset for the Unionida of the region spanning Italy and the coastal Croatian region (west of the Dinaric Alps). In total, 191 specimens were collected (85 Anodonta, 64 Unio, 17 Microcondylaea bonellii and 25 Sinanodonta woodiana) from 34 sites across the Italian Peninsula and coastal Croatian river basins for molecular identification (COI, 16S and 28S). Genetic analyses were performed to understand major phylogenetic and phylogeographic patterns. Seven species were detected: three Anodonta species (A. anatina, A. cygnea and A. exulcerata), two Unio species (U. mancus and U. elongatulus), Microcondylaea bonellii, and the invasive Sinanodonta woodiana. The presence of three endemic species (A. exulcerata, U. elongatulus and M. bonellii) confirms the importance of the region as a centre of endemism for freshwater mussels. The Apennine Mountains act as an important biogeographic barrier., This work was partially supported by the Portuguese Foundation for Science and Technology (FCT) under grants SFRH/BPD/108445/2015 (EF) and SFRH/BD/115728/2016 (MLL). Cindy Bogan reviewed a draft of this manuscript. We are grateful to the two anonymous reviewers for their helpful remarks and suggestions that improved the quality of the manuscript; and to the Go¨teborg Natural History Museum for the loan of Swedish tissue samples., info:eu-repo/semantics/publishedVersion