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The Louros Basin hosts one of the most important karst systems of Epirus Prefecture (Greece) and plays a key role in supplying three counties with drinking water. Aiming to investigate the origin of groundwater and its flow patterns, a multi-tracer approach was used to describe and evaluate the hydrogeology of the system. Therefore, 271 surface water and groundwater samples were collected and analyzed for physicochemical parameters, major ions, and trace and rare earth elements, as well as stable isotopes (δ
18
O and δ
2
H). These data provided meaningful tracing of the water origin, water–rock interaction processes, and relationships among the aquifers. In particular, the elaboration of the major ions supported by the distribution of rare earth elements indicated that there are three aquifers located at different levels hosted in the Senonian and Pantokrator limestone formations. These aquifers are hydraulically interconnected by a cascade and constitute the Louros karst system which is drained by the homonymous river. Hydrochemical and isotopic data revealed that the Louros karst system is isolated from the adjacent northern Ioannina Basin and it is being recharged by precipitation. Higher groundwater salinity, where present, is mainly associated with increased water–rock interaction due to longer and deeper hydrologic flow, favoring the dissolution of evaporitic, carbonate and phosphate minerals.