Chromolaena odorata (L.) R. M. King and H. Robinson (Asteraceae), originally from the Neotropics, has become a serious weed in the humid tropics and subtropics of Southeast Asia, Africa and Pacific Islands. In its introduced distributions, C. odorata has been recognised as two biotypes, the Asian/West African (AWA) biotype and South African (SA) biotype, with independent distribution, morphology and ecological characters. To characterise the genetic variability and identify the likely source regions in the native distributions of the two biotypes, we carried out an extensive phylogeographic study using chloroplast and nuclear DNA sequences and microsatellite DNA markers. The analysis of both DNA sequences and nuclear markers showed that native populations possessed high genetic diversity, while both the AWA and SA biotypes in invaded regions appeared to have low genetic diversity. The AWA and SA biotypes were genetically distinct. Strong competitive ability and environmental adaptability may have facilitated the invasion AWA and SA biotypes in its respective invasive regions. We conclude that the source of AWA biotype may be Trinidad and Tobago, while the SA biotype was from Cuba and Jamaica. For a better outcome of biocontrol, the potential biological control agents for the two biotypes should be collected from these native regions, respectively.