Abstract: Isoetes is a genus of primitive vascular plants classified in the subdivision Lycopsida and occupies a very important position in the evolutionary history of the pteridophytes. In recent years, Isoetes has declined rapidly in population quantity (number) and has even disappeared from many locations in Mainland China. The genus is now considered to be rare and threatened or endangered in China and is listed among first category protected plants. The progressive decline in populations of Isoetes in Mainland China has been attributed to on-going anthropogenic changes; however, no data have been adduced to support this assumption. In this study, all present locations of Isoetes populations and locations where the genus had been previously recorded but is now extinct were investigated in Mainland China. The habitat characteristics, including elevation, water pH value and the conditions of substrate were measured at each site. Associated plants growing together with Isoetes were collected from each population. The result indicated that the geographic distribution of Isoetes in Mainland China is discontinuous and that every population of Isoetes is insulated from other populations. The three species of Isoetes in Mainland China occur at different elevations. The current populations showed no obvious differences in water pH and other characteristics. But habitats where Isoetes plants had been extirpated currently have higher water and substrate pH values compared to the period when the sites supported Isoetes populations. These sites also had higher readings than the current locations of remaining Isoetes populations. This study indicates that populations of Isoetes growing in marshy areas close to farmland are faced with intense competition from other hydrophyte species whose proliferation is encouraged by the use of fertilizers in adjacent farmland. The populations of Isoetes growing in the freshwater intertidal zone avoid competition with other hydrophytes because of the periodic fluctuation of water level and thus grow comparatively well. The results indicate that the geographic distribution and occurrence of Isoetes plants in Mainland China is closely correlated with elevation, water pH, competition with other species, and water depth.