The northern fowl mite (NFM) Ornithonyssus sylviarum Canestrini and Fanzago is a blood-feeding ectoparasite found on many wild bird species and is a pest of poultry in the United States. It is unknown where NFM infestations of poultry originate, which has made it difficult to establish preventative biosecurity or effective control. We used microsatellite markers to evaluate genetic variation within and among NFM populations to determine routes of introduction onto farms and long-term persistence. We compared NFM from flocks of chickens (Gallus gallus) on different farms in California, Washington, and Georgia, and we compared NFM collected over a 5-yr interval. On three farms we collected NFM from chickens and house sparrows (Passer domesticus) nesting on each farm, which we used to assess movement between host species.There was strong genetic structure among mites from different poultry farms and low estimates of migration between farms. There were significant differences between mites on chickens and house sparrows on two farms where sparrows nested near flocks, indicating no exchange of mites. Only one farm showed evidence of NFM movement between chickens and sparrows.There was high genetic similarity between mites collected 5 yr apart on each of two farms, indicating that NFM infestations can persist for long periods.The genetic patterns did not reveal sources of NFM infestations on chicken farms.The data suggest that NFMs are strongly differentiated, which likely reflects periodic population declines with flock turnover and pesticide pressure.