Triangle sail mussel (Hyriopsis cumingii) is the most important mussel species commercially exploited for freshwater pearl culture in China. Its genetic diversity was studied among populations from the five largest freshwater lakes of China, Poyang Lake (PY), Dongting Lake (DT), Taihu Lake (TH), Hongze Lake (HZ), and Chaohu Lake (CH), by the polymorphism of the inter-simple sequence repeats (ISSRs). Polymerase chain reaction (PCR) detections showed that a total of 62 loci were amplified from eight primers; of those, 31.88%, 31.15%, 31.03%, 27.27% and 26.92%, respectively, were polymorphic in each of the five populations across all genotypes tested. The average heterozygosities were 0.4747, 0.3274, 0.2366, 0.2099 and 0.2018 in each of these populations from PY, TH, DT, CH and HZ, respectively. Phylogenic analyses showed that PY and TH populations clustered with CH and HZ and formed a group, while the DT population on its own formed a separated branch. The smallest distance (0.0404) was scored between PY and TH populations, indicative of their closest relationship; the biggest distance (0.2438) was found between PY and DT populations, suggesting their greatest divergence. The present study provided genetic basis for managing mussel stocks from these lakes separately to best preserve the genetic diversity of H. cumingii. On the other hand, since the population in PY displayed the highest genetic diversity, it may be used preferably in future selective breeding to improve pearl yield and quality.