Isoëtes is an ancient lycopod lineage with a highly conserved morphology that provides few morphological characters to resolve the phylogeny of its species. Species appear to have evolved by divergence and allopolyploidy. The basic diploids I. hypsophila, I. taiwanensis, and I. yunguiensis and the tetraploid I. sinensis occur in China. Analysis of ITS sequences indicates that the Chinese Isoëtes species are part of an Australasian clade including I. brevicula from Western Australia and I. kirkii from New Zealand. Two distinct cloned sequences of the second intron of a LEAFY homolog were recovered from I. sinensis supporting the hypothesis that I. sinensis is an allotetraploid. One of the I. sinensis cloned sequences was similar to the I. taiwanensis sequence and the other cloned sequence was similar to the I. yunguiensis sequence identifying I. taiwanensis and I. yunguiensis as the likely parents of I. sinensis. Other cloned sequences recovered from I. sinensis were recombined parts of the two distinct sequences. Morphological evidence supporting an allotetraploid origin of I. sinensis is found in its larger microspore size and intermediate megaspore texture compared to I. taiwanensis, and I. yunguiensis.