Alien species invasion is one of the most important drivers of worldwide environmental change, which may result in environmental degradation, biodiversity loss, and food and water shortage. It may also increase the possibility and severity of natural disasters, and damage international trade and benefits. In last two decades, DNA-based molecular markers were widely used to detect the genetic diversity of invaded alien species. Inter-simple sequence repeat (ISSR) is a microsatellite-based technique, with the superiorities of simple, quick, reliable, and generating higher levels of DNA polymorphism, and being used as a new molecular marker for genetic study. This paper introduced the principles, characteristics and procedures of ISSR, and summarized its applications in studying the genetic structure, genetic diversity, origin, distribution mode, phylogenesis, and breeding features of invasive plants.