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Recent phylogenetic reconstruction of the Aizooideae (Aizoaceae) revealed that it and several of its genera are polyphyletic. Whereas the previous phylogeny was only based on chloroplast markers, we utilize here both nuclear and chloroplast markers. The four chloroplast markers together produce a well-resolved and well-supported phylogenetic hypothesis, while many branches in the tree derived from the nuclear internal transcribed spacer (ITS) are unsupported. The position of Acrosanthes as sister to Mesembryanthemoideae + Ruschioideae and the close relationship of Tetragonia schenckii to Tribulocarpus (Sesuvioideae) is highly supported by both nuclear and chloroplast markers. We revise the classification of the Aizoaceae, redefining subfamilial and generic boundaries to maintain monophyly. A new fifth subfamily, the Acrosanthoideae subfam. nov., is created for Acrosanthes, an isolated sister-lineage to the Mesembryanthemoideae + Ruschioideae. It is the only subfamily endemic to mesic fynbos and all other subfamilies are mainly associated with the more arid karroid vegetation. Basal, shortly stipitate ovules and a xerochastic, parchment-like capsule are synapomorphies for the Acrosanthoideae. The Eurasian endemic annual, Aizoanthemum hispanicum, is not related to the southern African species of Aizoanthemum and is placed in a new genus, Aizoanthemopsis gen. nov. The southern African genera Galenia and Plinthus are embedded within Aizoon. These genera were separated by differences in the size of their flowers. Since these features are highly homoplasious, we place all three under an enlarged Aizoon, which can be recognized by capsules with reduced expanding tissue as well as leaves and/or stems that are covered with silvery hairs or hair-like or rounded papillae. The Aizooideae now consist of the genera Aizoanthemopsis, Aizoanthemum, Aizoon, Gunniopsis and Tetragonia, with a total of 116 species. Our results also have consequences in the Sesuvioideae, where the generic name Anisostigma is resurrected for Tetragonia schenckii. The two species of Tribulocarpus and the monotypic Anisostigma are sister to the other Sesuvioideae and are characterized by tubular flowers. Since we have synapomorphies for the clade consisting of Anisostigma + Tribulocarpus and for the remainder of the Sesuvioideae (i.e., circumscissile capsules and seeds completely enveloped by an aril), we recognize two tribes, Sesuvieae and Anisostigmateae tr. nov., within the Sesuvioideae. For the expanded concept of Aizoon and for Tetragonia, we propose a new subgeneric classification to reflect the major clades retrieved. In Aizoon, four of the six subgenera are new and for Tetragonia, two of the four subgenera are new. We provide keys to the subfamilies of the Aizoaceae, to the genera and subgenera of the Aizooideae and also to the tribes of the Sesuvioideae. Distribution maps are provided for the Aizooideae, Acrosanthoideae and for the little-known Anisostigma schenckii.