Classification of the tribe Cardueae in natural subtribes has always been a challenge due to the lack of support of some critical branches in previous phylogenies based on traditional Sanger markers. With the aim to propose a new subtribal delimitation, we applied a Hyb-Seq approach to a set of 76 Cardueae species representing all the subtribes and informal groups defined in the tribe, targeting 1061 nuclear conserved orthology loci (COS) designed for Compositae and obtaining chloroplast coding regions as byproduct of off-target reads. For the extraction of target nuclear data, we used two strategies, PHYLUCE and HybPiper, and 776 and 1055 COS loci were recovered with each of them, respectively. Additionally, 87 chloroplast genes were assembled and annotated. With the three datasets, phylogenetic relationships within the tribe were reconstructed under approaches of concatenation (using supermatrices as input for maximum likelihood analysis with RAxML) and coalescence (species tree estimated with ASTRAL based on the individual gene trees of each COS locus). The phylogenetic analyses of the nuclear datasets fully resolved virtually all nodes with very high support. Although nuclear and plastid tree topologies are highly congruent, they still present some incongruences, which are shortly discussed. On the basis of the phylogenies obtained, we propose a new taxonomic scheme of 12 monophyletic and morphologically consistent subtribes: Carlininae, Cardopatiinae, Echinopsinae, Dipterocominae (new), Xerantheminae (new), Berardiinae (new), Staehelininae (new), Onopordinae (new), Carduinae (redelimited), Arctiinae (new), Saussureinae (new), and Centaureinae. Another main key result of the study was the high resolution recovered at the backbone of the Cardueae tree, which led to obtain better inter-subtribal relationships. Using as tree base the nuclear HybPiper phylogeny, we updated the temporal framework for the origin and diversification of the tribe and subtribes. Overall, the power of Hyb-Seq is demonstrated to solve relationships traditionally suggested by morphology but never recovered with support using Sanger sequencing of a few DNA markers.