The euryhaline fish Cyprinodon variegatus variegatus (Cvv) is capable of tolerating ambient salinities ranging from 0.3 to 167 g l−1, but incapable of long-term survival in freshwater (< 2 mM Na+). However, a population of this species, now designated as a subspecies (Cyprinodon variegatus hubbsi; Cvh), has been isolated in several freshwater (0.4–1 mM Na+) lakes in central Florida for the past ~150 ky. We previously demonstrated that Cvh has a significantly higher affinity for Na+ uptake suggesting that it has adapted to its dilute freshwater environment. We here evaluate whether Cvh should be considered a separate species by characterizing pre- and post-zygotic isolation, Na+ transport characteristics of the two populations and their hybrids, and developing a molecular phylogeny of Cvv and Cvh populations in Florida using mtDNA sequence data. We found evidence of partial prezygotic isolation with Cvv females mating almost exclusively (89%) with con-specific males in choice mating experiments. Partial post-zygotic isolation was also observed with significant (59–89%) reductions in hatching success of hybrid embryos compared with con-specific embryos. Na+ uptake kinetics in hybrids (both Cvv x Cvh and Cvh x Cvv) bred and raised under common garden conditions were intermediate to Cvh (high affinity) and Cvv (low affinity) indicating that observed differences are genetically based. Similar observations were made with respect to short-term (96 h) survival of juveniles acutely transferred from 7 mM Na+ to a range of more dilute (0.1–2 mM Na+) freshwater. Finally, although phylogenetic analysis of Cvv and Cvh populations using mtDNA sequence for ND2 were unable to fully resolve a polytomy between Cvh and Cvv populations from northeastern Florida, these data do not falsify the hypothesis that Cvh is of monophyletic origin. Overall, the available data suggest that Cvh should be considered a separate species or at a minimum an evolutionarily significant unit.