The Florida Scrub-Jay (Aphelocoma coerulescens), a habitat specialist listed as Threatened by the U.S. Fish and Wildlife Service, exhibits extremely limited natal dispersal. We hypothesize that this behaviour results in a correlation of genetic and geographical distance. To test this, we are sampling intensively along the north–south orientated Lake Wales Ridge of central Florida. We describe the isolation and characterization of 31 microsatellite loci developed to determine the minimum distance at which genetic structure can be detected, and to test for isolation by distance. There are 3–15 alleles per locus, with expected heterozygosities from 0.16 to 0.86.