Isoëtes cipoensis and I. longifolia, two new endemic tetraploids from the Cerrado (Brazilian savanna), are described, illustrated, and compared to their similar species. Isoëtes cipoensis can be distinguished by a set of characters such as flaccid, straight, and erect leaves, deltate labia, presence of conspicuous sclerified phyllopodia, baculate megaspores with frequent long spine-like macroelements, and distally echinate microspores. Isoëtes longifolia is characterized by having a fully submerged habitat in the river, flaccid, long leaves (70–100 cm long), oblong labia, uniformly baculate megaspores, and densely echinate microspores. Importantly, chloroplast genomes already published for the species further support their phylogenetic distinctness in comparison to other species from the tropical region in Brazil. This study provides a key and distribution map for the species from the Brazilian savanna and scanning electron microscopy images of the mega- and microspores for the new species. These species are only known from their type location. We suggest I. cipoensis and I. longifolia should be classified as Data Deficient (DD) and Endangered (EN) species, respectively, based on the IUCN criteria.