Freshwater ecosystems are among the most severely affected environments by species loss caused by climate change and intense anthropogenic pressure. To preserve biodiversity, biomonitoring plays a key role by providing reliable data on biological diversity and ecological status. Environmental DNA (eDNA) metabarcoding has emerged as a powerful and non-invasive alternative to traditional morphology-based sampling and identification methods. This study represents the first application of eDNA analysis to assess the invertebrate communities in three Sicilian Lakes: Poma, Piana degli Albanesi and Scanzano. Water samples were collected at two points in each lake and after filtration with nitrocellulose membranes, eDNA was extracted and metabarcoding analysis was performed. A total of 27 species were identified, belonging to Phyla of Annelida, Arthropoda and Rotifera. Notably, the analysis revealed the presence of alien species (