The term hotspots of subterranean biodiversity has been used to define subterranean habitats with an arbitrary cutoff of twenty or more obligate stygobitic and troglobitic species. Until present, no hotspots of subterranean biodiversity had been identified in South America. Thus, the objective of this work is to present the first two hotspots of subterranean biodiversity in that continent. The two hotspots of subterranean biodiversity are the Toca do Gonçalo cave (22 spp.) and Areias cave systems (28 spp.). The cave species, some of them considered relict species, belong to the Platyhelminthes (1 sp.), Nemertea (1 sp.), Gastropoda (2 spp.), Amphipoda (2) Isopoda (7), Decapoda (1), Collembola (5), Coleoptera (5), Ensifera (1), Sternorrhyncha (1), Zygentoma (1), Diplopoda (6) Chilopoda (5) Araneae (2), Opiliones (1) Palpigradi (2), Pseudoscorpiones (4), and Osteicthyes (2). Although both caves, together, have 50 troglobitic species, only 38% of these species are formally described. Both caves have perennial water bodies, but terrestrial obligate cave invertebrates are dominant in number of species in both systems (around 77%). While the Areias system is partially contained in a conservation unit, Toca do Gonçalo cave is currently unprotected, although it certainly deserves protection.