In order to investigate the level of genetic diversity of the endangered species Brasenia schreberi, three populations from Jiangsu and Zhejiang Provinces were sampled and analyzed using inter-simple sequence repeats (ISSR) markers. A total of fifty-four individuals were sampled. Twelve filtrated from 77 ISSR primers gave rise to 101 discernible DAN fragments of which 16 were polymorphic (15.84%). The average number of DNA bands amplified by each primer was 8.4. The Nei's genetic and Shannon's information indexes were 0.0395 and 0.0630 respectively at species level, 0.0391 and 0.0335 respectively at population level. Based on Nei's Gst value and Shannon's index, it showed that most of genetic diversity (85.79%) was within populations, and that gene flow (Nm) was 3.0199. It was concluded that vegetative propagation, habitat loss and fragment, introducing plants, and human disturbances may play a significant negative role on genetic diversity and genetic structure of Brasenia schreberi.