Functional connectivity is a key factor for the persistence of many specialist species in fragmented landscapes. However, connectivity estimates have rarely been validated by the observation of dispersal movements. In this study, we estimated functional connectivity of a real landscape by modelling dispersal for the endangered natterjack toad (Bufo calamita) using cost distance. Cost distance allows the evaluation of ‘effective distances’, which are distances corrected for the costs involved in moving between habitat patches in spatially explicit landscapes. We parameterized cost-distance models using the results of our previous experimental investigation of natterjack's movement behaviour. These model predictions (connectivity estimates from the GIS study) were then confronted to genetic-based dispersal rates between natterjack populations in the same landscape using Mantel tests. Dispersal rates between the populations were inferred from variation at six microsatellite loci. Based on these results, we conclude that matrix structure has a strong effect on dispersal rates. Moreover, we found that cost distances generated by habitat preferences explained dispersal rates better than did the Euclidian distances, or the connectivity estimate based on patch-specific resistances (patch viscosity). This study is a clear example of how landscape genetics can validate operational functional connectivity estimates.