Analysis of the trnT-trnL spacer, the trnL intron, the trnL-trnF spacer, and the rps16 intron of 106 species in 34 of the 45 genera of New World Asclepiadeae in the former subtribes Metastelmatinae, Oxypetalinae, and Gonolobinae shows that these genera form a well-supported clade (“MOG”). The small Andean genus Pentacyphus is sister to the remaining MOG clade. The former Gonolobinae form a well-supported subclade closely related to members of Tassadia, Funastrum, and former Oxypetalinae. The only genera that are monophyletic as presently circumscribed are Tassadia and Funastrum. The separation of Tweedia from Oxypetalum is justified by our results. Philibertia is monophyletic only if Amblystigma, Fontellaea, Melinia, Mitostigma, and Podandra are also included. Most species of Blepharodon and Hemipogon are not monophyletic with the respective type species. Some former Astephanus and Cynanchum species as well as the monotypic Grisebachiella are imbedded in Diplolepis (D. descolei, D. geminiflora, D. hieronymi, D. boerhaviifolia, and D. nummulariifolia). The neglected tribe Orthosieae is recognized at subtribal level, Orthosiinae. Chromosome numbers (as far as known) indicate a tendency for reduction in several clades. This study provides a framework for future study of the American MOG genera and highlights areas that need further attention.