Despite its ancient origins, worldwide distribution, and adaptation to diverse habitats, Isoëtes species have a highly conserved morphology, making it difficult to resolve phylogenetic relationships using morphological characters. In this paper, we report the results from various analyses (maximum parsimony, maximum likelihood, and Bayesian inference) for Isoëtes species from around the world based on nucleotide sequences from the nuclear internal transcribed spacer (ITS) and chloroplast atpB/rbcL intergenic spacer regions. The trees resulting from our analyses of the combined data contain six major well-supported clades (bootstrap ≥ 90%, posterior probabilities = 1.00): A clade with possible Gondwanan affinities (I. australis, I. coromandelina, I. panamensis, I. cubana, I. jamaicensis); a South African clade (I. capensis, I. toximontana, I. stellenbossiensis, I. stephansenii); a largely Northern Hemisphere clade (I. nuttallii, I. orcuttii, I. minima, I. dixitei, I. abyssinica, I. olympica, I. longissima, I. velata); an Asian/Australasian clade (I. drummondii, I. gunnii, I. pusilla, I. kirkii, I. muelleri, I. taiwanensis, I. japonica, I. yunguiensis, I. habbemensis); a Mediterranean clade (I. histrix and I. setacea); and a poorly resolved clade consisting of 12 new world species (American species complex). Our results are compared to past classifications and various biogeographical scenarios are explored.