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Abstract Aim Although divergences in nuclear DNA content among different species within a genus are widely acknowledged, intraspecific variation is still a somewhat controversial issue. The aim of this study was to assess genome size variation in the polymorphic species Picris hieracioides L. (Asteraceae) and to search for potential interpretations of the size heterogeneity. Location Europe. Methods The genome sizes of 179 plants of P. hieracioides collected from 54 populations distributed across 10 European countries were determined by propidium iodide flow cytometry. Differences in nuclear DNA content were confirmed in simultaneous analyses. Results 2C-values (population means) at the diploid level varied from 2.26 to 3.11 pg, spanning a 1.37-fold range. The variation persisted even after splitting the whole data set into two recently distinguished morphotypes (i.e. the ‘Lower altitude’ type and the ‘Higher altitude’ type) that possess significantly different nuclear DNA contents. Cluster analysis revealed the presence of three major groups according to genome size, which exhibited a particular geographical pattern. Generally, the genome size of both morphotypes increased significantly from south-west to north-east. A new cytotype, DNA triploid, was found for the first time. Main conclusions High intraspecific variation in the amount of nuclear DNA in P. hieracioides correlates with the extensive morphological variation found within the taxon. Despite the complex pattern that was presented, genome size variants were non-randomly distributed and reflected palaeovegetation history. We suggest that the complex evolutionary history of P. hieracioides (e.g. the existence of several cryptic lineages with different levels of cross-interactions) is the most plausible explanation for the observed heterogeneity in genome size.