The Florida grasshopper sparrow, Ammodramus savannarum floridanus, is a non-migratory, endangered subspecies endemic to the prairie region of south-central Florida. It has experienced significant population declines and is currently restricted to five locations. We found substantial levels of variation in microsatellites and mtDNA control region sequences, estimates of inbreeding genetic effective population sizes that were much larger than the estimated census size, and no evidence of inbreeding within five sampled populations (n = 105). We also found a lack of genetic structure among populations (FST = 0.0123 for microsatellites and θ = 0.008 for mtDNA), and evidence for dispersal between populations, with 7.6% of all individuals identified as immigrants to their population of capture. We suggest that the subspecies be managed as a single management unit on a regional scale rather than as multiple management units on a local subpopulation scale. There is still a limited opportunity to preserve much of the present genetic variation in this subspecies, if immediate measures are taken to reverse the current population decline before this variation is reduced by genetic drift.