The lycophyte genus Isoëtes L. is nearly cosmopolitan, with approximately 350 species, 23 of which occur in Brazil. The lack of adequate distinguishing morphological characters in the leaves and stems, as well as the frequent cases of hybridization and polyploidy, makes the identification of species very difficult. Usually the spores provide the best characters for distinguishing species within Isoëtes, but these can vary in size because of polyploidy and hybridization. In this work we studied the variation in size of both mega- and microspores, and the relationship between size and ploidy level. We also present the first chromosome counts for seven species that are endemic to Brazil: I. goebelii (2n  =  33), I. martii (2n  =  44), I. maxima (2n  =  22), I. organensis (2n  =  66), I. ramboi (2n  =  44) I. smithii (2n  =  44), and I. spannagelii (2n  =  22). Our results suggest a correlation between ploidy level and spore size can be useful for the establishment of a preliminary hypothesis on polyploidy and hybridization among Brazilian Isoëtes.