Crofton weed, Ageratina adenophora,a troublesome weed in the world,is an invasive organism in China. Genetic diversity of thirty-two Chinese populations of A. adenophora from different areas was analyzed with the inter-simple sequence repeat (ISSR) technique. It indicated that its genetic diversity was rich. Nei's gene diversity and Shannon's information index were 0.235 and 0.372 at species level respectively. Genetic variation of Crofton weed mainly existed in the population, and about 34.5% of the total variance was attributable to population divergence and 65.5% to individual differences within populations. The Mantel Z-statistic test showed that the genetic distance between populations generally increased with geographic distance (r=0.542,p< 0.001),which indicated that isolation by distance is one of the blocks of gene flow of A. adenophora. Genetic diversity level of Crofton weed had the trend to decrease along with the increased altitude (r=0.368,P<0.001), and the mean of Nei's gene diversity and Shannon's diversity indices both decreased with the increased altitude.