We report the development of five microsatellite markers for the invasive North American native plant Ambrosia artemisiifolia (Asteraceae). An enrichment protocol was used to isolate microsatellite loci and polymorphism. was explored with samples from 16 natural populations collected in the native range, across North America, and in an invaded area along the Rhone Valley in France. In these areas, we found a high level of polymorphism within population and significant heterozygote deficiencies.