Taxonomic complexity has hindered the partitioning of the arti®cial genus Centaurea, even though it has long been recognized as a polyphyletic assemblage. On the basis of morphology, pollen type, karyology and DNA sequence analysis, previous workers have de®ned ®ve informal groups in the genus (Acrocentron, Centaurea sensu stricto, Cyanus, Jacea and Psephellus). However, the precise delimitation of these groups and their relationships remain largely unknown. Moreover, although some informal groups have been established among the rest of the subtribe (Amberboa, Carthamus and Stemmacantha), many genera cannot be classi®ed in any group. Newer molecular approaches are essential for resolving these problems. Sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA and the chloroplast gene matK were analysed for a comprehensive sample of the whole subtribe, with the aim of clarifying the delimitation and the phylogeny of the groups of the Centaureinae. Results largely con®rm the suggested informal entities as natural groups, with some interesting changes of placement of some genera, especially in the Acrocentron and the Stemmacantha groups. Our results con®rm that the sections of Centaurea with Dealbata pollen type should be classi®ed as a dierent genus, Psephellus. In addition to morphology and suggested pollen type evolution, the ITS and matK phylogenies are also supported by karyological evidence. Our results con®rm that the natural delimitation of Centaurea that minimizes nomenclatural changes is possible only if a new type of the genus is designated.