The annual killifishes of the genus Cynolebias Steindachner, 1876, include one of the well-supported clades composed of the Cynolebias adloffi species complex of the Ban˜ados del Este Biosphere Reserve Site, from the Atlantic coastal wetlands in Uruguay. One member of this clade, Cynolebias charrua, has been considered as an intergradation complex of populations between C. adloffi and C. viarius by showing high levels of morphological variability. Systematic affinities between both taxa have been historically discussed. In the present study, a phylogeographic approach based on mitochondrial cytochrome b haplotype analysis and chromosome data are presented. According to the present data a scenario of a multiple simultaneous speciation process and perhaps reticulation events is possible in the C. adloffi species complex, followed by divergence associated with Quaternary marine transgressions in this region. This hypothetic scenario could explain the remarkable genetic diversity and the lack of phylogenetic resolution among clades in C. charrua, as well as the disconcordance in the phylogenetic relationships among Cynolebias from different data sets. Several parameters of DNA polymorphism and chromosome data have also suggested that populations of C. charrua from southern Laguna Merin, in eastern Uruguay and southern Brazil, could represent a relictual group from a widespread ancestral population. Chromosome rearrangements like pericentric inversions in isolated populations could have triggered cladogenetic events from this highly polymorphic population. Present analysis suggests that the metapopulation could represent the management units in these endangered killifishes and that the populations of C. charrua from the Laguna Merin basin could be included in a high-priority conservation programme.