Genetic diversity can be used to describe patterns of gene flow within and between local and regional populations. The Florida Everglades experiences seasonal fluctuations in water level that can influence local population extinction and recolonization dynamics. In addition, this expansive wetland has been divided into water management regions by canals and levees. These combined factors can affect genetic diversity and population structure of aquatic organisms in the Everglades. We analyzed allelic variation at six DNA microsatellite loci to examine the population structure of spotted sunfish (Lepomis punctatus) from the Everglades. We tested the hypothesis that recurrent local extinction and recent regional divisions have had an effect on patterns of genetic diversity. No marked differences were observed in comparisons of the heterozygosity values of sites within and among water management units. No evidence of isolation by distance was detected in a gene flow and distance correlation between subpopulations. Confidence intervals for the estimated F-statistic values crossed zero, indicating that there was no significant genetic difference between subpopulations within a region or between regions. Notably, the genetic variation among subpopulations in a water conservation area was greater than variation among regions (Fsp>FPT). These data indicate that the spatial scale of recolonization following local extinction appears to be most important within water management units.