Giant reed (Arundo donax) is an invasive weed that is native to the Old World. Tens of thousands of hectares of riparian habitat in the Rio Grande Basin (RGB) in Texas and Mexico have been heavily affected by invasions of Arundo. Additionally, many other watersheds across the southwestern United States have also been affected. Giant reed is being targeted for biological control because it displaces native vegetation and consumes water that could potentially be used for agricultural and municipal purposes, especially in areas with limited access to water. Finding the best-adapted insects for biological control involves locating the origin(s) of this plant. To narrow down the proximal source(s) of invasion of giant reed in the RGB, 10 microsatellite markers were developed. An analysis of 203 Old World and 159 North American plants, with an emphasis on the RGB, indicated a reduction in the allelic diversity in the introduced range compared with the Old World. Clonal assignment, neighbor joining, principal coordinates analyses, and STRUCTURE analyses were consistent and implied multiple introductions in North America, with one (likely clonal) lineage responsible for the invasion of the RGB, northern Mexico, and other parts of the southwestern United States. Although no identical matches with the RGB lineage were found in the Old World, several close matches were found on the Mediterranean coast of Spain.