Location of river-groundwater exchange zones and biodiversity hotspot is essential for a river management plan, especially for Mediterranean karstic rivers. This location is often difficult and time-consuming when long river sectors are considered. We studied a 13 km-long sector of the Cèze River (Southern France) located in a karstic canyon. We compared five indicators of river-groundwater exchanges: longitudinal profiles of temperature, electrical conductivity and 222Rn concentrations in the surface water of the river, chemical characteristics of the hyporheic water and hyporheic biodiversity. Upwelling zones occurred downstream of geomorphological heterogeneities (here at the tail of gravel bars). Surface water chemistry, especially electrical conductivity and 222Rn concentrations, clearly traces large scale gaining sections, which were not associated to valley narrowing but with lateral springs, suggesting a crucial role of the geological structuration of the karstic plateau of Méjanne-le-Clap. Hyporheic water chemistry fits with the large-scale hydrological pattern, but with a high variability corresponding to local heterogeneities. The stygobite fauna (obligate groundwater organisms) and benthic EPTC (Ephemeroptera, Plecoptera, Trichoptera and Coleoptera) occurred preferentially in the gaining sections fed by groundwater, likely because of oligotrophic water and cooler temperature. The spatial distribution of river-groundwater exchange zone and hyporheic biodiversity may be thus predicted using changes in surface water chemistry, especially for electrical conductivity and 222Rn concentrations.