European calcareous grasslands have decreased dramatically in area and number during the last two centuries. As a result, many populations of calcareous grassland species are confined to small and isolated fragments, where their long-term survival is to some extent uncertain. Recently, several restoration projects have been initiated to enlarge the current grassland area in order to maintain the exceptionally high species richness. However, from a genetic point of view, the success of these restoration measures is not necessarily guaranteed, as strong historical decreases in population size and limited gene flow may have led to low genetic diversity through genetic bottlenecks and drift. In this study, we investigated genetic diversity and structure of 16 populations of the calcareous grassland specialist Cirsium acaule in a severely fragmented landscape in south-western Belgium. The overall distribution of this species in the study area was significantly and positively related to patch area, suggesting that small patches do not allow survival of this species. Both allelic richness and genetic diversity were significantly and positively related to population size. Estimation of observed and expected gene diversity provided evidence for population bottlenecks in the history of not less than 31% of all sampled populations. Reconstruction of the historical land use showed that patch area decline in populations that went through a recent bottleneck was significantly larger than that in populations that showed no evidence of a bottleneck. Assignment analyses showed low migration rates, suggesting that replenishment of lost alleles through gene flow is highly unlikely. Overall, our results indicate that in the absence of gene flow strong decreases in calcareous grassland area may have long-lasting effects on genetic diversity of plant populations and may hamper the success of restoration projects that simply aim at restoring initial habitat conditions or enlarging population fragments, as indicated by the fact that none of the recently restored areas has been occupied by C. acaule.