Changes in agricultural production methods over the last century have caused a massive reduction and fragmentation of the area of European semi-natural grasslands. It remains unclear whether small and isolated grassland fragments can support viable plant populations in a sustainable way. In our study area in southern Belgium, the extent of calcareous grasslands was reduced from c. 650 ha in 1775 to less than 30 ha in 2004. We used AFLP markers to quantify the effects of present and historical grassland fragmentation on the genetic structure of 27 populations of the rare perennial plant species Globularia bisnagarica. Given the mixed breeding system of the species and the relatively small area of the studied system, the populations were characterized by high genetic differentiation (F(st) range: 0.42-0.48; Phi(st)=0.53). A Mantel test revealed significant isolation by distance of the populations. Average within population genetic diversity, measured as expected heterozygosity or gene diversity, was low (H(j) =0.081) and was negatively related to population isolation. This suggests more gene flow into less isolated populations. Population size and local habitat characteristics did not significantly influence population genetic diversity. Both, high selfing rates in G. bisnagarica and a population genetic response to habitat fragmentation may explain our findings. Finally, a clear geographical clustering was observed, with cluster membership partially explainable by historical grassland connectivity. If populations indeed started to differentiate after fragmentation, this process was not (yet) strong enough to erase the genetic similarity between fragments that historically belonged to the same large grassland fragment.