Isoetes (Isoetaceae, Isoetales) is a cosmopolitan genus with an ancient and diverse evolutionary history that presumably peaked in the Palaeozoic. The age of the living clade has never been satisfactorily clarified. With little morphological (and molecular) divergence among species, megaspore morphology may provide valuable information on relationships in the group, not least among extant and extinct forms. We study megaspore ornamentation and surface texture/structure in 74 phylogenetically placed samples representing 59 species of Isoetes using scanning electron microscopy, and we discuss evolutionary implications of the results. Ornamentation (classified into 12 categories) and surface structure/texture (10 categories) were mapped onto a phylogeny constructed based on molecular data from the same samples whenever possible. All megaspores of the family are trilete with an outermost siliceous coating. There is substantial micromorphological variation, of which some appears clade specific. The megaspores of Isoetes reticulata, discovered from the late Oligocene to early Miocene of Tasmania, share remarkable similarities with those of extant species in the Australasian clade, in particular Isoetes neoguineensis and partly also Isoetes japonica. Adding a minimum age of the Australasian crown group (clade D) based on this fossil improved log marginal likelihood values of dating analyses significantly, and the fossil may provide key information for the understanding of divergence times of clades in the Isoetaceae crown group.