Isoetes amazonica and I. gardneriana were the first two species of the genus to be collected from Brazil. Isoetes amazonica was gathered by Richard Spruce in the Amazon basin near Santarém in the state of Pará in 1850. Isoetes gardneriana was collected by George Gardner in the current Dianópolis in Tocantins State in 1843. Despite being known for a long time by botanists, these species have not been recollected since then, which raised questions about their taxonomic recognition, current distribution ranges and conservation status. Fieldwork efforts led to the rediscovery of I. amazonica and I. gardneriana after 167 years. These collections enrich our understanding of their habitats and morphologies. We provide here re-descriptions for these species. Based on IUCN criteria, Isoetes amazonica and I. gardneriana should be assigned as data deficient (DD) and endangered (EN), respectively. The rediscovery of these species raises hopes that other areas in Amazon and Cerrado biomes harbour I. amazonica and I. gardneriana , respectively. This study will serve as a basis towards the conservation of these species.