Species diversity and intraspecific genetic diversity play a critical role in conservation and restoration of grassland ecosystems. To maintain regional adaptations of native wild plants, seeds for restoration projects are produced regionally. The delineation of regions is organised by seed transfer zones (STZs). Generalised STZs that apply uniformly to many species have been established in several European countries. Ideally, generalised STZs should be based on comprehensive data of intraspecific genetic and phenotypic diversity for a larger number of species. However, such underlying data is missing. The project RegioDiv aims to fill this gap and generate empirical data on genetic variation of multiple grassland plant species across Germany. Here we describe the driving principles and main methods of the project. A total of 33 species were collected at an average density of ∼1 sample/1000 km² across the 22 existing STZs, and a total of 11,976 samples were genotyped with SNP markers. The analysis of genetic population structure included cluster analysis and analyses of isolation-by-distance and isolation-by-environment. An exemplary within-species analysis for Agrostis capillaris, a widespread grass, revealed five intraspecific genetic clusters, distributed in spatially coherent ranges that did not fully match the STZs. Most of the STZs differed genetically following a pattern of isolation-by-distance and isolation-by-environment. In an across-species analysis, genetic differentiation was affected by mating system and ploidy. Outcrossed and polyploid species were less differentiated than self-compatible and diploid species. However, genetic differentiation did not significantly differ between grasses and herbs, highlighting the variability among species within these groups. The dataset of the RegioDiv project will advance both basic and applied research on genetic variation of grassland plant species. The results will allow the assessment of the current German STZ system and guide potential improvements.