The species composition and molecular phylogeny of Senecio sect. Jacobaea (Asteraceae; Senecioneae) were studied to identify the closest relatives of Senecio jacobaea. Maximum parsimony and Bayesian inference analyses of DNA sequence data of the plastid (the trnT-L igs, the trnL intron, two parts of the trnK intron, and the psbA-trnH igs) and nuclear genome (ITS1, 5.8S, and ITS2) showed these markers to be suitable to assess the species composition of sect. Jacobaea, identifying 24 species as members of this section. Of these, nine species were not previously assigned to the section. The selected DNA sequence regions, however, showed too little sequence divergence to be optimal for phylogenetic inference within sect. Jacobaea. In contrast, AFLPs proved to be too variable to be used to study relationships between the basal lineages in sect. Jacobaea. Nonetheless, these markers are very useful to study the phylogeny of S. jacobaea and its closest relatives. The combined use of DNA sequence data and AFLPs allowed us to take a major step towards resolving phylogenetic relationships in sect. Jacobaea, identifying Senecio alpinus, Senecio pancicii and Senecio subalpinus (using DNA sequence data) or Senecio chrysanthemoides (using AFLPs) as the closest relatives of S. jacobaea.