Many cooperatively breeding birds exhibit fine-scale spatial genetic structure as a result of restricted dispersal and habitat specialization. Sitta pusilla (Brown-headed Nuthatch) is a cooperatively breeding bird restricted to mature pine-dominated forests of the southeastern United States and has been undergoing population declines across most of its range. We used five polymorphic microsatellite loci developed for this species to examine fine-scale spatial genetic structure within a site in northern Florida as well as broader genetic structure among this site and two other sites (a second in northern Florida and one in southern Georgia). Spatial autocorrelation analyses within the more densely sampled site detected positive spatial genetic autocorrelation up to 1300 m in males when auxiliary males were included, but no autocorrelation was found in females or in males when auxiliary males were excluded. At the broader scale, we found small but significant genetic differentiation among all three populations, including two sites that were separated by less than 40 km of suitable habitat. Our results suggest that both sexes of the Brown-headed Nuthatch exhibit limited dispersal, with philopatric male auxiliaries contributing to more pronounced genetic structure over small geographic distances compared to females. Our sampled populations were in a region where much suitable habitat remains, yet we still observed limited dispersal. This finding suggests that in more fragmented regions, populations may become isolated and at risk of extinction. © 2010 Humboldt Field Research Institute. Abstract reproduced by permission.