Background To set up successful conservation measures, detailed knowledge on the dispersal and colonization capacities of the focal species and connectivity between populations is of high relevance. We developed species-specific nuclear microsatellite molecular markers for the grayling (Hipparchia semele), a butterfly endemic to Europe and of growing conservation concern in North-West Europe, and report on its population genetics, in a fragmented, anthropogenic landscape in Belgium. Our study included samples from 23 different locations nested in two regions and additional historical samples from two locations. We assessed contemporary, long-distance dispersal based on genetic assignment tests and investigated the effect of habitat loss and fragmentation on the population genetic structure and genetic variation using data of nine microsatellite loci. Results Detected dispersal events covered remarkably long distances, which were up to ten times larger than previously reported colonisation distances, with the longest movement recorded in this study even exceeding 100 km. However, observed frequencies of long-distance dispersal were low. Our results point to the consequences of the strong population decline of the last decades, with evidence of inbreeding for several of the recently sampled populations and low estimates of effective population sizes (Ne) (ranging from 20 to 54 individuals). Conclusions Our study shows low frequencies of long-distance dispersal, which is unable to prevent inbreeding in most of the local populations. We discuss the significance for species conservation including future translocation events and discuss appropriate conservation strategies to maintain viable grayling (meta) populations in highly fragmented, anthropogenic landscapes.