Gonolobus (Apocynaceae, Asclepiadoideae) is a New World genus comprising an estimated 100–150 species. Variation in estimated species numbers is largely the result of still poorly known tropical taxa and differences regarding generic limits. Characters historically used to delimit genera such as Gonolobus within Gonolobinae—such as laminar dorsal anther appendages and various follicle morphologies—have been controversial and their evolution remains unknown, not having been explored in a phylogenetic framework. The primary objectives of the current study were to (1) test the monophyly of Gonolobus sensu Woodson in the context of a phylogeny of New World Asclepiadeae and (2) explore the evolution of laminar dorsal anther appendages and winged follicles with respect to their potential utility in generic circumscription. Chloroplast (trnL–F, rpsl6) data are newly presented for sixty-three taxa of Gonolobinae, representing an increased sampling of the subtribe from a maximum of seven taxa in prior studies. These data were combined with a previously published dataset to form a 183 taxa matrix of New World Asclepiadeae. Nuclear (LEAFY) data were newly generated for forty-nine taxa of Gonolobinae. Evidence from parsimony and Bayesian analyses of chloroplast and nuclear data presented here supports the monophyly of both the subtribe and the genus Gonolobus in a narrow or broad sense. Laminar dorsal anther appendages are restricted to Gonolobus s.l. or s.s., although parsimony remains equivocal on whether they evolved once or twice. A transversion in the trnL–F spacer is shown synapomorphic for Gonolobus s.s. Two indels in LEAFY, as well as winged follicles, are shown synapomorphic for Gonolobus s.l.