Taxonomic complexity has hindered the partitioning of the artificial 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 defined five informal groups in the genus ( Acrocentron , Centaureasensu 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 classified 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 mat K 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 confirm 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 confirm that the sections of Centaurea with Dealbata pollen type should be classified as a different genus, Psephellus . In addition to morphology and suggested pollen type evolution, the ITS and mat K phylogenies are also supported by karyological evidence. Our results confirm that the natural delimitation of Centaurea that minimizes nomenclatural changes is possible only if a new type of the genus is designated. Copyright 2001 Annals of Botany Company