Premise: Southern Africa is a biodiversity hotspot rich in endemic plants and lichen-forming fungi. However, species-level data about lichen photobionts in this region are minimal. We focused on Trebouxia (Chlorophyta), the most common lichen photobiont, to understand how southern African species fit into the global biodiversity of this genus and are distributed across biomes and mycobiont partners. Methods: We sequenced Trebouxia nuclear ribosomal ITS and rbcL of 139 lichen thalli from diverse biomes in South Africa and Namibia. Global Trebouxia phylogenies incorporating these new data were inferred with a maximum likelihood approach. Trebouxia biodiversity, biogeography, and mycobiont–photobiont associations were assessed in phylogenetic and ecological network frameworks. Results: An estimated 43 putative Trebouxia species were found across the region, including seven potentially endemic species. Only five clades represent formally described species: T. arboricola s.l. (A13), T. cf. cretacea (A01), T. incrustata (A06), T. lynniae (A39), and T. maresiae (A46). Potential endemic species were not significantly associated with the Greater Cape Floristic Region or desert. Trebouxia species occurred frequently across multiple biomes. Annual precipitation, but not precipitation seasonality, was significant in explaining variation in Trebouxia communities. Consistent with other studies of lichen photobionts, the Trebouxia–mycobiont network had an anti-nested structure. Conclusions: Depending on the metric used, ca. 20–30% of global Trebouxia biodiversity occurs in southern Africa, including many species yet to be described. With a classification scheme for Trebouxia now well established, tree-based approaches are preferable over “barcode gap” methods for delimiting new species.