Isoetes sinensis is a critically endangered aquatic quillwort, occurring in two fragmented sites of China (Xiuning county of Anhui; Jiande county of Zhejiang). During a five-year period (2004–2009), the areas and sizes of the two populations diminished dramatically due to intensive human activities. Genetic structure of the species was investigated using simple sequence repeat makers (SSRs). For seven nuclear microsatellites, high levels of genetic diversity were found within populations (HE = 0.324–0.447). Strong genetic differentiation was detected between populations (GST = 0.376), while weak genetic differentiation was found within populations (GST = 0.026–0.080). Notably, in contrast to the source–sink model suggested by previous study (Chen et al., 2009), the migration pattern of I. sinensis along the Xin’an River is best explained by the linear symmetrical non-adjacent flow model (LSNF), which indicates that intensive human activities of recent years have greatly affected the gene exchange pattern among I. sinensis subpopulations.