Abstract: To study the population genetics of Thielaviopsis basicola at molecular level, 54 isolates of T. basicola were collected from Chongqing, Gansu, Guizhou, Henan and Yunnan, and the genetic diversity and population differentiation among geographic populations were analyzed by ISSR method. The results showed that the average of bands amplified by 5 ISSR primers pairs was 9.6(8-12)and the average of polymorphism bands was 3.6(2-5). The Nei's index h, Shannon index I, G_ST value and the gene flow N_m of total populations were 0.350 7, 0.521 5, 0.338 9 and 0.975 6, respectively. The genetic distances of the 5 populations ranged from 0.113 4 to 0.275 2. The genetic identities between Henan and the other populations were higher with close genetic distances. The genetic identity of Guizhou population with Henan population was higher(0.804 8), with the other populations was lower. NTsys 2.10e cluster analysis indicated that the genetic similarity coefficients of different isolates were from 0.82 to 1.00 and the isolates could be clustered into 5 groups at the genetic similarity coefficient of 0.84. In conclusion, T. basicola maintains rich genetic diversity in China, there exists genetic differentiation among different populations, and the gene flow in populations was large.