Tribal delimitation of Cardueae is controversial, and the traditional classification in four subtribes (Echinopsidinae, Carlininae, Carduinae, and Centaureinae) has fluctuated widely. Most of the problems are centered in subtribes Echinopsidinae and Carlininae, often segregated with tribal rank. We therefore analyzed DNA sequences of the internal transcribed spaces (ITS) of the nuclear ribosomal DNA genes and the matK gene of the chloroplast DNA of a broad representation of the tribe to examine (1) the phylogeny of the tribe, (2) the position of Echinopsidinae and Carlininae, (3) the circumscription of the subtribes and the position of some conflicting genera, and (4) the delimitation of some generic complexes in the Carduinae. Phylogenetic analysis of ITS and matK sequence variation, both separate and combined, strongly support the monophyly of Cardueae including Carlininae and Echinopsidinae. The combination of both genomes suggest that Xeranthemum and its allies should be included among the Echinopsidinae rather than the Carlininae, which implies that the capitulum of Xeranthemum could be interpreted as a syncephaly. The subtribe Centaureinae forms a well-supported clade, and their sister clades contain the genera Arctium, Cousinia, Jurinea, and Saussurea from the Carduinae. However, some problems persist: Carduinae are a paraphyletic assemblage, and the subtribal placement of Berardia, Cardopatium, Cousiniopsis, and Staehelina remains unresolved. Our results also indicate that present classification in four subtribes is unsatisfactory, but it is still the only practical approach.