Many threatened plant species have genetic structures indicating effects of fragmentation, and in Central Europe arable plants are among the most threatened taxa. Although this threat has developed only recently in the course of agricultural intensification, their annual life form makes arable plants relatively sensitive to increasing fragmentation and decreasing population sizes. One of Central Europe's rarest arable plant species is Bupleurum rotundifolium (Apiaceae). To analyze the genetic structure and diversity of this species we sampled 27 populations of B. rotundifolium in Central Europe, and assessed genetic structure by amplified fragment length polymorphism (AFLP) fingerprinting. Ordination, clustering and also Bayesian analysis suggested that most of the populations of the Eastern part of Germany formed one cluster, and most of the Western German populations as well as populations from outside Germany built another group. The two clusters accounted for 24% of differences in genetic structure of B. rotundifolium populations, while there was relatively strong differentiation among (41% variance) and within populations of a given group (35% variance). The overall φ _ST-value was very high (0.65) and there was evidence for isolation-by-distance. Values of genetic diversity were very low for B. rotundifolium. The proportion of polymorphic loci per population varied between 9.4% and 38.7%, with those from eastern Germany being significantly less diverse (mean 19.1% vs. 25.5%).