The Lycophyta species are the extant taxa most similar to early vascular plants that were once abundant on Earth. However, their distribution has greatly diminished. So far, the absence of chromosome-level assembled lycophyte genomes has hindered our understanding of evolution and environmental adaption of lycophytes. We present the reference genome of the tetraploid aquatic quillwort, Isoetes sinensis, a lycophyte. This genome represents the first chromosome-level assembled genome of a tetraploid seed-free plant. Comparison of genomes between I. sinensis and Isoetestaiwanensis revealed conserved and different genomic features between diploid and polyploid lycophytes. Comparison of the I. sinensis genome with those of other species representing the evolutionary lineages of green plants revealed the inherited genetic tools for transcriptional regulation and most phytohormones in I. sinensis. The presence and absence of key genes related to development and stress responses provide insights into environmental adaption of lycophytes. The high-quality reference genome and genomic analysis presented in this study are crucial for future genetic and environmental studies of not only I. sinensis but also other lycophytes.