We report the first ever nation-wide study of the occurrence of non-indigenous species in Danish harbours. The sampling was car-ried out using both conventional and biomolecular methods (eDNA). In total, 16 harbours were covered – Esbjerg and Aarhus, the two largest harbours in Denmark, with intensive sampling and 14 harbours with a reduced programme. 26 non-indigenous species were recorded using conventional sampling and 13 species were recorded using eDNA-based methods. Excluding overlapping rec-ords, we have recorded a total of 34 non-indigenous species in the 16 harbours studied. Based on the results, we conclude the following: 1) more non-indigenous species are found in the western parts of Denmark (North Sea region) then in the eastern parts (Baltic Sea), and 2) a few species previously unseen in Danish marine waters were recorded, i.e. the two bristle worms Eteone het-eropoda (fam. Phyllodocidae) and Streblospio benedicti (fam. Spionidae). Further, we provide a proof-of-concept regarding the overarching objectives of the MONIS 1-3 projects and the eDNA-based test systems developed. The results constitute a baseline for future studies in Danish ports and other hotspot areas.