ABSTRACT A molecular phylogeny of the subfamily Ambavioideae (Annonaceae) was reconstructed using up to eight plastid DNA regions (matK, ndhF, and rbcL exons; trnL intron; atpB-rbcL, psbA-trnH, trnL-trnF, and trnS-trnG intergenic spacers). The results indicate that the subfamily is not monophyletic, with the monotypic genus Meiocarpidium resolved as the second diverging lineage of Annonaceae after Anaxagorea (the only genus of Anaxagoreoideae) and as the sister group of a large clade consisting of the rest of Annonaceae. Consequently, a new subfamily, Meiocarpidioideae, is established to accommodate the enigmatic African genus Meiocarpidium. In addition, the subfamily Ambavioideae is redefined to contain two major clades formally recognized as two tribes. The tribe Tetramerantheae consisting of only Tetrameranthus is enlarged to include Ambavia, Cleistopholis, and Mezzettia; and Canangeae, a new tribe comprising Cananga, Cyathocalyx, Drepananthus, and Lettowianthus, are erected. The two tribes are principally distinguishable from each other by differences in monoploid chromosome number, branching architecture, and average pollen size (monads). New relationships were retrieved within Tetramerantheae, with Mezzettia as the sister group of a clade containing Ambavia and Cleistopholis.