Divergent natural selection has the potential to drive the evolution of reproductive isolation. The euryhaline killifish Lucania parva has stable populations in both fresh water and salt water. Lucania parva and its sister species, the freshwater L. goodei, are isolated by both prezygotic and postzygotic barriers. To further test whether adaptation to salinity has led to the evolution of these isolating barriers, we tested for incipient reproductive isolation within L. parva by crossing freshwater and saltwater populations. We found no evidence for prezygotic isolation, but reduced hybrid survival indicated that postzygotic isolation existed between L. parva populations. Therefore, postzygotic isolation evolved before prezygotic isolation in these ecologically divergent populations. Previous work on these species raised eggs with methylene blue, which acts as a fungicide. We found this fungicide distorts the pattern of postzygotic isolation by increasing fresh water survival in L. parva, masking species/population differences, and underestimating hybrid inviability.