To examine the relationships of geographically isolated paleo-island populations of Pogonomyrmex badius (Latreille 1802) in Florida we generated a phylogeographic hypothesis based on mitochondrial DNA (mtDNA) sequences. We found at least three distinct mtDNA lineages and a positive correlation between genetic and geographic distances. The relationships between nowadays isolated P. badius populations might resemble a long lasting separation due to either restricted gene flow caused by inbreeding, paleo-climatic events or the impact of novel invasive species. The current depletion of the only representative of the ant genus Pogonomyrmex in the south-eastern USA makes a more fine-scaled mapping of the remaining, small P. badius populations necessary to identify evolutionary distinct units for conservation purposes.