To provide an overview of New World Asclepiadoideae, we here evaluate Asclepiadeae classification by comparing the taxonomic arrangement of subtribes with a topology obtained through analyses of two plastid DNA regions (trnL intron and trnLF intergenic spacer) for 111 species of Asclepiadoideae representing the major lineages of the subfamily. Without Jobinia, Nephradenia and Barjonia, Asclepiadeae are not monophyletic. The monotypic African genus Eustegia, with pendent pollinia, may represent the sister clade of Marsdenieae Ceropegieae, the group composed of plants with erect pollinia. Metastelmatinae including African plants are also nonmonophyletic, and the circumscription of the recently reinstated Cynanchinae should be studied further. Overall, Asclepiadeae are composed of three main clades. The Old World Astephaninae are the sister group of the other Asclepiadeae, which are divided into the ACTG (Asclepiadinae, Cynanchinae, Tylophorinae and Glossonematinae) and MOG (Metastelmatinae, Oxypetalinae and Gonolobinae) clades. According to this study, the New World Asclepiadoideae fall into just four clades: (1) Marsdenia (Marsdenieae), (2) Asclepias (Asclepiadinae, Asclepiadeae), (3) Cynanchum subgenus Mellichampia (Cynanchinae, Asclepiadeae), and (4) MOG, the clade comprising the majority of New World Asclepiadoideae.