Infrastructure is one of the main causes of landscape fragmentation, which results in isolation and loss of populations. Although the negative effect of roads on insects is well documented, only a minority of studies has focused on roads in the context of barriers to dispersal. Flying species in particular have been neglected. We investigated the effect of a four-lane motorway as a barrier to the movement of an isolated population of the threatened dragonfly Sympetrum depressiusculum in an agricultural landscape in Central Europe. Generalized additive models were used to assess the motorway's effect on (i) the distribution of adult dragonflies in patches of terrestrial habitat surrounding their natal site, and (ii) individual flight behaviour (i.e. willingness or unwillingness to cross the motorway). Movement patterns of marked adults throughout the landscape were also investigated. During one season, significantly fewer adults were found at patches located on the far side of the motorway, indicating it has a barrier effect. Observations on flight behaviour revealed no apparent effect of the motorway. The possible barrier effect for the species studied was therefore presumed to be a consequence of road mortality. Our results indicate that the motorway may influence the dispersal of this threatened species of dragonfly, which is a habitat specialist with particular requirements for its terrestrial environment. Negative effects on other species with similar behaviour and strategy can be presumed. When establishing new habitats, carrying out reintroductions or translocations, it is necessary to consider that roadways may reduce population size and affect population dynamics by limiting dispersal., Hana Šigutová, Filip Harabiš, Michal Hykel, Aleš Dolný., and Obsahuje bibliografii