The study investigates the genetic differentiation among two subspecies of Allium ursinum L., namely A. ursinum subsp. ursinum and subsp. ucrainicum as well as their putative hybrid that is represented by individuals with intermediate morphology. Inter-Simple Sequence Repeats (ISSR) were applied to determine the status of intermediate morphotypes in terms of their genetic pattern and to assess the level of genetic variability within and between various populations of A. ursinum. The study comprises 144 specimens from nine populations along the east-west transect in Poland, which includes localities of both subspecies and their putative hybrid. Among the examined populations, 48 bands were amplified, of which 45 were found to be polymorphic. The principal coordinate analysis (PCoA), the neighbour-net analysis and Mantel test showed a strong correlation between genetic variability and geographic distance. Analysis of molecular variance (AMOVA) revealed that a greater proportion of total genetic variation resided within populations rather than among them. The Structure Bayesian clustering analysis revealed the presence of three distinct genetic groups within studied populations, where 'eastern' genotypes correspond to A. ursinum subsp. ucrainicum, and 'western' to subsp. ursinum; whereas the third genetic group has the largest share in the individuals occurring at the border of the distribution ranges of both subspecies. The emergence of the third genetic group is probably an effect of hybridization events occurring within the secondary contact zone. Typical morphologically intermediate populations occur only in a relatively narrow geographical zone, but the hybrid zone revealed by molecular markers is actually much wider than it is suggested by the morphological pattern of individuals. The current distribution pattern of both subspecies of A. ursinum and their hybrid zone is related to the two main directions of postglacial migration of Fagus sylvatica to the area of Poland. The hybrid zone arose as an effect of the secondary contact of two divergent lineages of A. ursinum.K Ke ey y w wo or rd ds s: : Distribution pattern, genetic variability, hybrid zone, intermediate morphotype, ISSR.