Shallow water bodies dominate the areal extent of continental waters and host a proportion of biodiversity higher than the percentage of Earth’s surface they cover. Daphnia is a key component of small aquatic ecosystems food webs. Here we present the result of a survey in 24 ponds located in the core of Po river Basin, to assess the actual spreading of Daphnia species in one of the most productive areas of the Northern hemisphere. By using diagnostic genetic markers (12S rRNA and ND5 genes) we identified five Daphnia species: D. ambigua, D. curvirostris, D. longispina, D. obtusa and D. pulex in fourteen ponds. Additional analyses of two nuclear genes (LdhA and Rab4) revealed that D. pulex in the study area is native European strain. In opposite, D. ambigua shared haplotype with the North-Eastern American lineage that was introduced to Europe by long-distance dispersal. In the Po river Basin we identified a highly divergent lineage of D. longispina group that formed a clade with individuals from northern European Russia and might represent a new Daphnia species. Daphnia species in the Cremona province have European origin, except for D. ambigua which is a North American species spreading across Europe. Future attention will require monitoring of invasive species, particularly D. ambigua and the North American invasive clone of D. pulex that is already present in Northern Italy.