Theoretical studies suggest that mechanisms underlying habitat and population structure are important for shaping inter- and intraspecific variation in dispersal behaviour. Empirical evidence, especially in organisms living in spatially structured populations, however, is scarce. We investigated the relation between habitat configuration (patch size, connectivity) and dispersal by studying variation in tiptoe behaviour in the dune wolf spider, Pardosa monticola, under standardized laboratory conditions. Tiptoe behaviour prepares spiderlings for ballooning and can hence be considered as a precursor of aerial dispersal. The proportion of individuals that displayed tiptoe behaviour was highest in offspring from grasslands in a large dune landscape where habitat was continuously available, intermediate in offspring originating from a fragmented landscape, and lowest in offspring originating from a small and extremely isolated grassland patch. At the level of the fragmented landscape, variation was related to size and connectivity of four subpopulations. Both between and within landscapes, maternal condition had no effect on offspring dispersal. These results indicate that changes in habitat configuration from a large, connected landscape towards a small, fragmented one may lead to a decrease in dispersal rates, even at small spatial scales. Hence, behavioural traits narrowly linked to dispersal evolve towards less mobile phenotypes in small, isolated habitats, indicating high dispersal costs and low efficacy for gene flow in a spider species restricted to fragmented habitats.