Species-rich calcareous grasslands in Europe strongly declined during the twentieth century due to drastic land use changes. Many grasslands were converted into more productive pastures or are covered by shrubs or forests today, since they were overgrown after abandonment or afforested. Restoration of calcareous grasslands by shrub or forest clearing and subsequent recolonization of grassland species from adjacent grasslands is, therefore, an important conservation approach. Restored populations of calcareous grassland species may, however, differ from their source populations in genetic diversity and differentiation due to potential founder and bottleneck effects. In our study we analyzed, therefore, the impact of restoration by forest clearing and natural recolonization on the genetic variation of three common calcareous grassland species (Agrimonia eupatoria, Campanula rotundifolia, and Knautia arvensis) without a contribution of persistent seed bank, in South Western Germany. We used molecular markers AFLPs (Amplified fragment length polymorphisms) to compare genetic diversity within and differentiation between spontaneously recovered subpopulations with adjacent historically old, natural subpopulations at eight study sites. Restored parts of the grasslands have been re-established during the 1990s. Molecular markers revealed broadly similar levels of genetic diversity in source and restored subpopulations of the study species. Only A. eupatoria exhibited slightly higher diversity in restored subpopulations, which may be explained by higher dispersal potential due to the hooky fruits of the species. Genetic differentiation between source and restored subpopulations was not significant, indicating strong gene flow between the subpopulations. Our study underlines, therefore, that restoration of calcareous grasslands by natural recolonization after forest clearing is an efficient method to re-establish genetically variable subpopulations comparable to their sources.