Abstract: In coastal ecosystems, non-indigenous species (NIS) are recognized as a major threat to biodiversity, ecosystem functioning and socio-economic activities. Due to its geographic position and intensive maritime traffic, Portugal is particularly vulnerable to the introduction of marine NIS. The detection of an invasive species soon after its introduction, when the population is still confined to a small area and at a low density, maximises the probability of eradication or effective local management. To this end, the NIS-DNA project aims to optimize and implement DNA metabarcoding for NIS detection and monitoring in Portuguese coastal regions, including the archipelagos of Azores and Madeira. Under the NIS-DNA project we have been: i) assessing the gaps of NIS in reference sequences databases (ca. 30% of PT NIS still miss DNA barcodes); ii) generating DNA barcodes for missing species; iii) optimizing several steps of the metabarcoding workflow, namely the sampling strategies (i.e., bulk organismal samples, zooplankton, seawater eDNA), the DNA extraction protocols and the target genetic markers, and iv) detecting and monitoring NIS in recreational marinas along the Portuguese coast. So far, we have detected and identified 32 invertebrate NIS in marinas of the North of Portugal (namely crustaceans, molluscs, ascidians and bryozoans), 8 of which were first records, still pending visual confirmation. A key observation in these first results was the occurrence of very little overlap in the NIS detected either among sample types or between genetic markers. This implies that efficient metabarcoding-based monitoring of NIS requires the employment of at least two genetic markers (i.e., COI and 18S) and multiple sample matrices. Furthermore, because in our approach we targeted not only NIS, but all taxa within a given sample, the information retrieved on the taxonomic composition of the communities can be further explored. Namely, it can be used both for investigating the susceptibility of native communities to NIS introduction, and, on the other hand, if the NIS become invasive, to closely monitor their impact in the native communities. A more accurate and faster methodology will allow to develop an early warning system and to improve the monitoring programs of management frameworks, for helping to control the spread of NIS in Portuguese coastal ecosystems.