Plasmids have been a concern in the dissemination and evolution of antibiotic resistance in the environment. In
this study, we investigated the total pool of plasmids (plasmidome) and its derived antibiotic resistance genes
(ARGs) in different compartments of urban water systems (UWSs) in three European countries representing
different antibiotic usage regimes. We applied a direct plasmidome approach using wet-lab methods to enrich
circular DNA in the samples, followed by shotgun sequencing and in silico contig circularisation. We identified
9538 novel sequences in a total of 10,942 recovered circular plasmids. Of these, 66 were identified as con-
jugative, 1896 mobilisable and 8970 non-mobilisable plasmids. The UWSs’ plasmidome was dominated by small
plasmids (≤10 Kbp) representing a broad diversity of mobility (MOB) types and incompatibility (Inc) groups. A
shared collection of plasmids from different countries was detected in all treatment compartments, and plasmids
could be source-tracked in the UWSs. More than half of the ARGs-encoding plasmids carried mobility genes for
mobilisation/conjugation. The richness and abundance of ARGs-encoding plasmids generally decreased with the
flow, while we observed that non-mobilisable ARGs-harbouring plasmids maintained their abundance in the
Spanish wastewater treatment plant. Overall, our work unravels that the UWS plasmidome is dominated by
cryptic (i.e., non-mobilisable, non-typeable and previously unknown) plasmids. Considering that some of these
plasmids carried ARGs, were prevalent across three countries and could persist throughout the UWSs com-
partments, these results should alarm and call for attention.