Litter meadows, historically established for litter production, are species-rich and diverse ecosystems. These meadows drastically declined during the last decades along with decreasing litter use in modern livestock housing. The aim of our study was to identify the drivers of genetic variation in litter meadow species. Therefore, we tested whether genetic diversity and differentiation depend on habitat age, landscape structure, habitat quality, and/or population size. We analysed 892 individuals ofAngelica sylvestris,Filipendula ulmaria, andSuccisa pratensisfrom 20 litter meadows across the Allgau in Baden-Wurttemberg (Germany) using AFLP analyses. All study species showed moderate levels of genetic diversity, while genetic differentiation among populations was low. Neither genetic diversity nor differentiation were clearly driven by habitat age. However, landscape structure, habitat quality as well as population size revealed different impacts on the genetic diversity of our study species. Past and present landscape structures shaped the genetic diversity patterns ofA. sylvestrisandF. ulmaria. The genetic diversity ofF. ulmariapopulations was, moreover, influenced by the local habitat quality.S. pratensispopulations seemed to be affected only by population size. All explanatory variables represent past as well as present gene flow patterns by anthropogenic land use. Therefore, we assume that genetic diversity and differentiation were shaped by both historical creation of litter meadows via hay transfer and present mowing with agricultural machines. These land use practices caused and still cause gene flow among populations in the declining habitats.