Isoëtes cangae and Isoëtes serracarajensis (Isoetaceae) are species endemic to Carajás (Pará State, Brazil) that grow on iron-rich substrates of the Amazon basin under a seasonal rainfall regime. In addition to a few distinctive morphological traits, the species differ in terms of habitat and geographical distribution. The influence of the seasonality and habitat characteristics on quillwort morphology is poorly understood. To assess this influence, specimens from one population of I. cangae, and four populations of I. serracarajensis, were sampled in dry and rainy seasons to investigate variation among vegetative and reproductive characters. The megasporangium color, megaspore ornamentation, alae length, velum length and coverage (%), morphometry of sporangial wall cells, and polar/equatorial diameter ratio of megaspores provide consistent characters for species delimitation. The presence of stomata in I. serracarajensis is a new discovery and is related to its amphibious habit. Some characters that have been used in Isoëtes species descriptions, such as the number of leaves, leaf length, sporangium size, number of megaspores, and megaspore diameter were found to be variable. The current findings provide further information on the biology of the species and show the influence of seasonality and habitat characteristics on the morphological diversity of these lycophytes.