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The investigated area, located in the inner part of the Marche region (central Italy) and belonging to the carbonate Umbria- Marche ridges in the central Apennines, is characterised by very complex geo-structural setting and widespread karst phenomena that make difficult the definition of the relation among the aquifers basing only on the hydrogeological survey. Hence, the presence of different flowpaths among aquifers of the Umbria-Marche hydrostratigraphic sequence and of tectonic contacts among the different structures is verified using tracer tests. In particular, the tests showed that the Calcare Massiccio and the Maiolica aquifers are connected under certain tectonic conditions. A new tracer given by a single stranded DNA molecule and traditional fluorescent dyes have been injected into the Montelago sinkhole in different periods (during the recharge and during the discharge) and recovered in several points along the expected hydrogeological basin, using either manual and automatic sampling. Fluorescent traps were positioned in creeks, rivers and springs. The DNA molecule is useful to trace surface water and groundwater, is detectable even at very low concentrations, no significant change in water density and viscosity can be observed and its use is not dangerous for the environment. The results stress the suitability of DNA as hydrogeological tracer, capable to identify connections among aquifers and study different flowpaths even in high flow conditions when traditional tracers are more and more diluted. Moreover, fluorescein tracer allowed for the transport parameter determination, giving mean velocities ranging from 100 to 3000 m/day and mean residence time from some tens to hundreds of hours, and determining the aquifer volumes.