Genetic variability within and among fragmented populations of Artemisia pancicii was investigated in order to obtain a general understanding of the genetic structure related to the successful protection of this highly endangered species. Genetic variation within and among 15 populations of A. pancicii in Central Europe was analysed using amplified fragment length polymorphism (AFLP) and sequencing of two chloroplast DNA regions. The resulting polymorphism of AFLP loci was interpreted using basic population genetic indices and statistical visualisation. The total genetic variability within the populations was high (Ht = 0.248) and a highly differentiated population pattern (Fst = 0.241) was revealed. An analysis of molecular variance (AMOVA) revealed high variation among the populations (82%). There was no significant correlation between the genetic and geographic distance matrices. This indicates that population relatedness is not reflected in their geography. This was also confirmed by cpDNA sequencing. Highly restricted gene flow among the populations and genetic drift has resulted in reduced genetic variability in the smaller and highly differentiated A. pancicii populations, and very probably implies the presence of self-incompatibility and prevalence of clonal reproduction. The conservation of genetic variability in A. pancicii requires the persistence of large and also of small populations (because of population differentiation). The most important factor for the preservation of this species in the localities studied is the application of appropriate conservation management (such as mowing, grazing or fire management).