Background: Dactylis glomerata (orchardgrass or cocksfoot) is a forage crop of agronomic importance comprising high phenotypic plasticity and variability. Although the genus Dactylis has been studied quite well within the past century, little is known about the genetic diversity and population patterns of natural populations from geographically distinct grassland regions in Europe. The objectives of this study were to test the ploidy level of 59 natural and semi-natural populations of D. glomerata, to investigate genetic diversity, differentiation patterns within and among the three geographic regions, and to evaluate selected populations for their value as genetic resources. Results: Among 1861 plants from 20 Swiss, 20 Bulgarian and 19 Norwegian populations of D. glomerata, exclusively tetraploid individuals were identified based on 29 SSR markers. The average expected heterozygosity (HE,C) ranged from 0.44 to 0.59 and was highest in the Norwegian region. The total number of rare alleles was high, accounting for 59.9% of the amplified alleles. 80.82% of the investigated individuals could be assigned to their respective geographic region based on allele frequencies. Average genetic distances were low despite large geographic distances and ranged from D = 0.09 to 0.29 among populations. Conclusions: All three case study regions revealed high genetic variability of tetraploid D. glomerata within selected populations and numerous rare and localized alleles which were geographically unique. The large, permanent grassland patches in Bulgaria provided a high genetic diversity, while fragmented, semi-natural grassland in the Norwegian region provided a high amount of rare, localized alleles, which have to be considered in conservation and breeding strategies. Therefore, the selected grassland populations investigated conserve a large pool of genetic resources and provide valuable sources for forage crop breeding programs.