The inter-simple sequence repeat(ISSR) was conducted to analyze the genetic variation of four native populations of Trachidermus fasciatus in China - Zhejiang population(Z), Shangdong population(S), Hebei population(H) and Yalvjiang Yalujiang population(Y), with 15 polymorphic primers selected from 100 ISSR primers. For each population, 30 samples were used in the study. Six to seventeen bands were amplified for each primer. Altogether, 181 useful loci were detected and out of them, 136 ones were polymorphic, accounting for 75.14%. Among the four populations, H population had the lowest effective allele number (1.258 5), Nei’s gene diversity(0.153 0), Shannon’s information index(0.232 2), and polymorphic loci percentage(49.17%), while Y population’s indexes (1.479 4, 0.277 7, 0.411 2, and 74.03%, respectively) were the highest. The corresponding values of Z and S populations were higher than those of H population and lower than those of Y population. Taking the four populations as a whole, the total Nei’s gene diversity and Shannon’s information index were 0.269 1 and 0.400 9, respectively, showing a relatively high diversity of T. fasciatus. The values of G_ST and N_m were 0.131 3 and 3.309 4, indicating medium differentiation and some degree of gene exchange that existed among the populations. As results of AMOVA indicated, there were 85.54% variation that existed in the individuals and 14.46% variation among populations. At the same time, there was significant differentiation among the four populations (P<0.05 or P < 0.01), too. In the UPGMA tree conducted on the genetic distance, Z and S populations clustered firstly, then with Y population, and finally joined to H population. Obviously, the Z, S, and Y populations had a relative close relationship according to their geographic distance, whereas H population showed clear divergence from the other three populations.