In the present study, inter-simple sequence repeats (ISSRs) markers were used to examine the genetic variability of Schistosoma japonicum isolates from different provinces in mainland China, using S. japonicum from Japan and S. mansoni from Puerto Rico for comparison. Of the 30 primers screened, 4 produced highly reproducible ISSR fragments. Using these primers, 107 discernible DNA fragments were generated with 105 (98.13%) being polymorphic, indicating considerable genetic variation among the examined S. japonicum isolates. The percentage of polymorphic bands among S. japonicum isolates from mainland China and Japan was 82.24%, 43.93% among mountainous type isolates and 64.49% among lake/marshland type isolates from mainland China. UPGMA analysis revealed that all of the S. japonicum samples were grouped into two clades, the first contained isolates from mainland China, and the other one contained samples from Japan. Within the cluster of S. japonicum isolates from mainland China, isolates from mountainous Sichuan and Yunnan provinces grouped together, whereas isolates from lake/marshland regions (Anhui, Jiangsu and Hubei provinces) clustered together. The results of present study demonstrated that the ISSR markers are useful for studying genetic diversity and population structure of S. japonicum isolates from mainland China.