The Eupatorium adenophorum is one of the most widespread invasive alien species in China. In the present study, the genetic variation and population structure of this species were analyzed using amplified fragment length polymorphism (AFLP) markers. Nine primer pairs were selected for the analysis and 685 bands were produced, among which 474 bands were polymorphic (PPB = 69.2%). Diversity levels within populations were relatively high (mean expected heterozygosity = 0.188, mean Shannon index = 0.296). Regression analysis showed a significant positive relationship between Shannon genetic diversity and altitude (R2 = 0.31). However, there was a negative correlation between Shannon genetic diversity and latitude (R2 = 0.16), as well as between Shannon genetic diversity and longitude (R2 = 0.45). Cluster analysis grouped the majority of the weed populations into three main clusters that corresponded with the geographic regions. At the regional level, the AMOVA indicated that about 70% of the variations in the data set were from genotypic variations within populations, 13.3% of the variations were due to regional differences, and the remaining 16.6% were due to differences among populations within the provincial regions. The results imply that most individuals tested in the present study should have been produced through seeds, and the process of colonization resulted in progressive loss of genetic diversity from the southwest to the northeast of China. Key words: Amplified fragment length polymorphism, invasive species, Eupatorium adenophorum, diversity, genetic variation, population structure