Аннотация:The Ponto-Caspian region is an endemicity hotspot that harbours several crustacean radiations,among which amphipods are the most diverse. These poorly known species are severely threatenedin their native range, while at the same time they are invading European inland waters withsignifcant ecological consequences. A proper taxonomic knowledge of this fauna is paramount forits conservation within the native region and monitoring outside of it. Here, we assemble a DNAbarcode reference library for nearly 60% of all known Ponto-Caspian amphipod species. We use severalmethods to defne molecular operational taxonomic units (MOTUs), based on two mitochondrialmarkers (COI and 16S), and assess their congruence with current species-level taxonomy based onmorphology. Depending on the method, we fnd that 54–69% of species had congruent morphomolecular boundaries. The cases of incongruence resulted from lumping distinct morphospecies intoa single MOTU (7–27%), splitting a morphospecies into several MOTUs (4–28%), or both (4–11%).MOTUs defned by distance-based methods without a priori divergence thresholds showed the highestcongruence with morphological taxonomy. These results indicate that DNA barcoding is valuablefor clarifying the diversity of Ponto-Caspian amphipods, but reveals that extensive work is neededto resolve taxonomic uncertainties. Our study advances the DNA barcode reference library for theEuropean aquatic biota, paving the way towards improved taxonomic knowledge needed to enhancemonitoring and conservation eforts.