Mexican sunflower is a native species of North and Central America that was introduced into China early last century, but it has widely naturalized and become a harmful invasive plant in tropical and subtropical regions in South China. Inter-simple sequence repeat (ISSR) markers were employed to assess genetic diversity and variation in Mexican sunflower populations from China and neighboring regions. The karyotypes of populations were also studied. Our research showed high levels of genetic diversity in all populations. The lowest genetic diversity estimates were represented in two populations in Laos, suggesting prevention of new introductions into Laos is critical. Partitioning of genetic variance revealed that genetic variation was mostly found within populations, and unweighted pair group method with arithmetic means (UPGMA) analysis showed that the introductions into China and Laos were independent. There were no obvious correlations between genetic relationships and geographic distance of populations in China, consistent with the human associated dispersal history of Mexican sunflower. Previous cytological data and our chromosome count (2n = 34) and karyotype analysis showed chromosome stability among populations. The high levels of genetic diversity within invasive Mexican sunflower populations could be challenging for its management in China, and further expansion and potential negative effects on ecological systems of this plant should be monitored.