The management of biological invasions, which pose a growing threat to natural resources and human well-being, is critical for reducing associated negative impacts. As part of the process of developing a strategy for the management of biological invasions in the South African National Biodiversity Institute’s (SANBI) gardens, we collated a list of alien plant species from 13 gardens as part of a situational analysis. We requested lists of alien plant species recorded in each of the SANBI’s gardens. A total of 380 records included 225 alien plant species belonging to 73 families. A significant number of species were intentionally introduced through horticultural trade as ornamentals (49%; n = 225), while 20.9% were consumed as either food or medicine by humans. Plant life forms included woody and herbaceous plants, graminoids, succulents and ferns. Herbaceous (42.7%; n = 225) and woody plants (3.8%) were the dominant life forms. The Walter Sisulu National Botanical Garden had the highest number of alien species (88 species), followed by Kirstenbosch (61 species) and Pretoria (46 species) National Botanical Gardens, with herbaceous species constituting the largest number in all gardens (i.e., 47, 19, and 27 species, respectively). The number of species that we recorded that were listed in the National Environmental Management: Biodiversity Act (NEM: BA) (Act No. 10 of 2004): Alien and Invasive Species Regulations’ categories were not notably different from the number of unlisted species (58.2% vs. 42.8%). The number of species listed in the different categories varied significantly across the different gardens, with a significantly higher number of unlisted species and of Category 1b species in the Walter Sisulu, Kirstenbosch and Pretoria National Botanical Gardens than in other gardens. That a significantly larger number of alien species originated from South America points to the need to improve biosecurity controls on existing relations. The results of this study provided a baseline database to help comparison between successive surveys in future.