Isoetes are ancient quillworts members of the only genus of the order Isoetales. The genus is slow evolving but is resilient, and widespread worldwide. Two recently described species occur in the Eastern Brazilian Amazon, Isoetes serracarajensis and Isoetes cangae. They are found in the ironstone grasslands known as Canga. While I. serracarajensis is present mostly in seasonal water bodies, I. cangae is known to occur in a single permanent lake at the South mountain range. In this work, we undertake an extensive morphological, physiological and genetic characterization of both species to establish species boundaries and better understand the morphological and genetic features of these two species. Our results indicate that the morphological differentiation of the species is subtle and requires a quantitative assessment of morphological elements of the megaspore for diagnosis. We did not detect differences in microspore output, but morphological peculiarities may establish a reproductive barrier. Additionally, genetic analysis using DNA barcodes and whole chloroplast genomes indicate that although the plants are genetically very similar both approaches provide diagnostic characters. There was no indication of population structuring I. serracarajensis. These results set the basis for a deeper understanding of the evolution of the Isoetes genus.