Polyploidy plays an important role in the speciation of Isoëtes. Increasing our knowledge about the specific origin of each polyploid or phylogenetic relationship among species has been hampered because of conserved morphological variation and scarce habitats. We present several hypotheses concerning the speciation pathways of Isoëtes species distributed in East Asia. Our hypotheses are inferred from phylogenetic relationships that were elucidated using sequences of the internal transcribed spacer regions of nuclear ribosomal DNA, a second intron of LEAFY, and chloroplast DNA trnS-psbC spacer regions. These inferred phylogenetic relationships indicated that (1) the Chinese tetraploid, I. sinensis, is closely related to I. yunguiensis; (2) the Korean endemic species, I. hallasanensis, is an autotetraploid derived from I. taiwanensis or closely related taxa; (3) the hexaploid I. coreana forms a clade and has its closest evolutionary relationships with I. taiwanensis or I. hallasanensis; and (4) the Japanese hexaploid I. japonica is closely related to I. taiwanensis-I. coreana and I. sinensis-I. yunguiensis. These results suggest that interspecific hybridization and polyploidization have played central roles in speciation of East Asian Isoëtes. Furthermore, I. taiwanensis, an endemic species in Taiwan, has been involved in at least three cases of autopolyploid or allopolyploid speciation in East Asia.