Fishes that tolerate salinities above 60 ppt (hypersaline saltwater) appear to use largely similar, but exaggerated, mechanisms as during standard seawater acclimation (~32-35 ppt). One unique aspect of hypersaline saltwater acclimation is further gill cell junction tightening to retain water while maintaining high paracellular Na+ export. The upregulation of “hypersalinity-specific” tight junction paralogs (i.e., claudin 10c and 10f) has been associated with this change in gill permeability in the Common Killifish (Fundulus heteroclitus). Here, we further test the hypothesis that these paralogs contribute to high hypersaline saltwater tolerance in F. heteroclitus by comparing their responses to the Banded Killifish (F. diaphanus), a less-tolerant congener, and their natural F1 hybrids (F. diaphanus × F. heteroclitus). As predicted, proxies of iono- (plasma Na+, Cl- and K+) and osmoregulatory homeostasis (muscle water content) suggest F. heteroclitus has a higher tolerance to 60 ppt than F. diaphanus, while F1 hybrids have intermediate tolerance. At the molecular level, only F. heteroclitus significantly upregulated total relative Claudin 10c and 10f mRNA content in the gill after 24 hours at 60 ppt. To study Claudin 10c regulation, we measured allele-specific expression in F1 hybrids. The mRNA content of the F. heteroclitus allele matched F. diaphanus at 10 ppt, but was 2-fold higher at 60 ppt, suggesting that cis-regulatory evolution has led to the hypersalinity-induced increase of Claudin 10c in F. heteroclitus. These data support the hypothesis that hypersalinity specific, cation selective tight junction paralogs contribute to the evolution of extreme hypersalinity tolerance in fishes.