[Aim] To study the genetic diversity and structure of Ambrosia artemisiifolia and Ambrosia trifidain different geographical populations. [Method] A total of 240 samples from eight A. artemisiifolia populations and seven A. trifida populations were labeled with 13 ISSR primers. [Result] A total of 304 bands were amplified from 128 samples from the A. artemisiifolia populations. We observed 98.68% polymorphic loci. Shannon's information index and Nei's gene diversity index were 0.6716 and 0.4788, respectively. A total of 266 bands were amplified from 112 samples from the A. trifida populations. We observed 95.86% polymorphic loci. Shannon's information index and Nei's gene diversity index were 0.6593 and 0.4670, respectively. The genetic distances of A. artemisiifolia and A. trifida populations were similar. [Conclusion] ISSR analysis showed that A. artemisiifolia and A. trifida had genetic stability among their populations, but with rich genetic diversity within the population. The genetic variations in A. artemisiifolia and A. trifida mainly occurs within the population. There was a significant correlation between genetic and geographical distances of A. artemisiifolia populations, but there was no significant correlation between those of the A. trifida populations.