A molecular phylogenetic investigation was carried out to clarify aspects of the systematics of Oxylobus, a primarily Mexican alpine genus of Eupatorieae. Analysis of sequence data from two nuclear (nrDNA ITS, ETS) and three plastid markers (rbcL, ndhF, matK) confirmed the monophyly of Oxylobus and placed species of Ageratina as its sister group. A survey of 56 samples of Oxylobus using nrDNA ITS and ETS provided support for the currently accepted species, and showed the recently described O. coyulensis to be distinct and the sister group to the rest of the genus. The results also confirmed the placement of O. juarezensis in synonymy with O. subglabrus. The results of a broad survey of Ageratina for ITS data showed that it is likely not monophyletic as currently circumscribed. The phylogenetic results also highlighted the distinctiveness of Piqueria and Piqueriopsis as a distinct clade at the base of Eupatorieae.