DNA metabarcoding has the capacity to bolster current biodiversity assessment techniques, including the early detection and monitoring of non-indigenous species (NIS). However, the success of this approach is greatly dependent on the availability, taxonomic coverage and reliability of reference sequences in genetic databases, whose deficiencies can potentially compromise species identifications at the taxonomic assignment step. In this study we assessed lacunae in availability of DNA sequence data from four barcodes (COI, 18S, rbcL and matK) for NIS occurring in European marine and coastal environments. NIS checklists were based on EASIN and AquaNIS databases. The highest coverage was found for COI for Animalia and rbcL for Plantae (up to 63%, for both) and 18S for Chromista (up to 51%), that greatly increased when only high impact species were taken into account (up to 82 to 89%). Results show that different markers have unbalanced representations in genetic databases, implying that the parallel use of more than one marker can act complimentarily and may greatly increase NIS identification rates through DNA-based tools. Furthermore, based on the COI marker, data for approximately 30% of the species had maximum intra-specific distances higher than 3%, suggesting that many NIS may have undescribed or cryptic diversity. Although completing the gaps in reference libraries is essential to make the most of the potential of the DNA-based tools, a careful compilation, verification and annotation of available sequences is fundamental to assemble large curated and reliable reference libraries that provide support for rigorous species identifications.