The aim of this study is to assess the spatio-temporal variability of lateral flows in the streams of a large karstic basin to construct a conceptual model of karst contributions to flood generation. The lateral flows of the Tarn River, which crosses the Grands Causses karst zone in Southern France, were investigated between several gauging stations along the river. First, through analysing the lateral flows on an event time scale of 30 floods, it was possible to identify the losing and gaining reaches, highlighting a highly variable attenuation/amplification role of karsts on flood generation. Second, the diffusive wave model was used to quantify the lateral flows on an hourly time step within a flood event. The simulations show a high variability of lateral outflows and inflows within a same reach according to the hydrometeorological conditions, with in some cases an inversion of the lateral flow direction during the flood. The results highlight complex surface/groundwater exchanges during a single flood event, with high river losses despite the concurrent flow of large springs feeding the stream. This spatio-temporal variability of the karst influence on flood generation was linked to the aquifer’s structure, which has improved the understanding of the hydro(geo)logical functioning of the Grands Causses massif. Finally, the new methodology proposed here opens challenging perspectives towards a framework for the analysis of surface–groundwater exchanges in karstic rivers.