Abstract: The genus Isoëtes is a widely distributed group of lycophytes. This paper reports on the discovery of a new population of Isoëtes in Yunnan Province, China The population is hexaploid and contains 66 chromosomes. The spores were observed using scanning electron microscopy, which revealed several unique characteristics. Notably, the megaspores (diameter 470 – 500 µm, mean of 480 µm) exhibited a reticulate ornamentation on the proximal and distal surfaces, while the microspores displayed an echinate pattern to their surfaces. These characteristics distinguish the new species from the previously documented hexaploid species in China, I. orientalis Hong Liu & Q. F. Wang. Phylogenetic analysis based on chloroplast genome data further supported recognition of this distinct species. Thus, we identified and named the new species I. fengii Y. F. Gu & Y. H. Yan, sp. nov. in the present paper. Morphologically, diploid species I. yunguiensis Q. F. Wang & W. C. Taylor shares similarities in leaf and megaspore characters with the new species but differs by laevigate-granulate microspores and ovate sporangia. I. fengii sp. nov. also differs from the hexaploid species I. japonica A. Braun, previously thought to be distributed in China and characterized by laevigate microspores. Because of their limited geographic range, small population size, low number of individuals, and disturbed habitats, I. fengii sp. nov. is evaluated as critically endangered (CR). To aid in the identification and conservation of these rare and endangered plants, we provide a key to all currently known quillworts in China.