Secondary freshwater fish species inhabiting fluctuating and extreme environments are susceptible to changes in dispersion, effective population size, and genetic structure. The Iberian toothcarp Aphanius iberus is an endemic cyprinodontid of the Iberian Peninsula restricted to brackish water of salt marshes and coastal lagoons on the eastern Spanish Mediterranean coast. In this study, we analyzed mitochondrial cytochrome b (cyt b) DNA and microsatellite variation to evaluate ways in which the processes of extinction, dispersal, and colonization of A. iberus across its geographic distribution have affected its population genetic structure over time and space. The A. iberus network reconstruction indicated subtle levels of phylogeographic structuring. This, combined with substantial mitochondrial DNA (mtDNA) genetic diversity, suggests that Pleistocene glaciations had a lesser effect on the demographic structure of its populations than was the case for Iberian freshwater species with a similar distribution. Haplotype network, hierarchical analysis of molecular variance, and pairwise ΦST comparisons involving some Levantine samples showed a relatively high degree of mtDNA differentiation, which could be explained by historical isolation of the Villena Lagoon population. Conversely, significant genetic differentiation that follows an isolation-by-distance pattern, and a reduction in Ne though time was detected with microsatellites, suggesting extensive habitat fragmentation on the Mediterranean coast of the Iberian Peninsula over the past hundreds of years. At a smaller geographical scale (Mar Menor Lagoon), habitat fragmentation, probably due to human activity, appears to have resulted in substantially reduced migration and increased genetic drift, as shown by expanded genetic differentiation of populations.