Neotropical migrant birds have undergone significant changes in population size, due in part to alterations in the landscape in their breeding range. Some alterations have resulted in the isolation of areas containing optimal habitat for breeding. Isolation between habitat fragments could reduce gene flow between populations, which might result in loss of genetic variation due to genetic drift. To test whether isolation due to distance between populations may affect migratory birds as much as sedentary birds, we used four microsatellite loci as genetic markers to compare gene flow and population structure in migratory and sedentary House Wrens (Troglodytes aedon and T. musculus, respectively). If migratory behavior enhanced gene flow, we expected to find that populations of migratory birds were genetically more similar than populations of sedentary birds, and that gene flow declined more rapidly with distance in sedentary than in migratory populations. Blood samples were collected from 18–25 migratory House Wrens at each of six sites in Ohio separated by distances from 25 to over 300 km, and from 16–20 sedentary House Wrens at each of six sites in Costa Rica, also at distances up to 350 km. We used heterologous microsatellite primers to obtain estimates for R_ST (population subdivision) and Nm (number of immigrants per generation) in relation to distance between pairs of populations. In the migratory populations, Mantel permutational tests showed no effect of distance on R_ST or Nm at distances between 25 and 300 km. In the sedentary birds, R_ST increased and Nm decreased significantly as distance between populations increased from 25 to 300 km. F_ST values were not different from those obtained using Slatkin's (1995) R_ST statistics. These results are consistent with our hypothesis that migratory behavior enhances gene flow.