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Onosma is a species-rich genus with complicated patterns of morphological and karyological variation, and controversial taxonomic treatments. In the present study we focused on the Asterotricha group, one of three major infrageneric groups, in the area of central and southern Europe. Ribosomal internal transcribed spacer (ITS) sequences and amplified fragment length polymorphism (AFLP) markers were used to assess species differentiation and relationships, and to gain insight into the evolutionary history of the group. ITS data supported the monophyly of the asterotrichous taxa. Six genetically differentiated entities were resolved by AFLP markers, corresponding to O. echioides, O. heterophylla s.l., O. thracica including O. rigida, O. malkarmayorum, O. taurica, and O. stellulata. Onosma stellulata is identified as the most differentiated lineage within the Asterotricha group, which is consistent with its unique chromosome number. Within O. heterophylla s.l., recognition of O. viridis as a species distinct from O. heterophylla s.str. is supported. On the other hand, O. tornensis appears to be conspecific with O. viridis. Inferring from the patterns of genetic diversity and divergence, the evolutionary history of O. heterophylla s.l. is discussed. Genetic variation within the polymorphic Apennine-Dalmatian O. echioides is almost continuous and shaped by isolation by distance, thus none of the previous intraspecific taxonomic concepts are valid. The assumed hybrid origin of O. malkarmayorum is supported, and its affinity to potential ancestors, O. heterophylla s.l. and O. thracica incl. O. rigida, is indicated. Future studies of asterotrichous Onosma species should focus on diversity centers in the eastern Mediterranean and Asia Minor, to build up a comprehensive taxonomic concept of the group and to clarify its evolution. [ABSTRACT FROM AUTHOR]