Variation in lighting environments creates different demands of visual systems for the successful detection and interpretation of visual signals. Eye size is a critical property of the visual system as it has strong effects on visual acuity and visual sensitivity. While many comparative studies have examined eye size across fishes that live in disparate lighting environments (i.e., caves versus surface habitats, mesopelagic versus pelagic depths, turbid versus clear water, diurnal versus nocturnal), fewer have investigated differences in eye size as a function of water clarity at the among-population level. Here, we compared relative eye size (eye size residuals on standard length) among wild-caught Bluefin Killifish (Lucania goodei) from tannin-stained swamps and clear springs across four drainages in Florida. We also performed a laboratory rearing experiment where we reared animals in clear and tea-stained water, which mimic spring and swamp conditions, to determine whether phenotypic plasticity as a function of lighting conditions influences relative eye size. Field caught animals varied greatly in relative eye size among populations, but there was no clear relationship with lighting environment. Fish from the two southern drainages (Everglades, Withlacootchee) had greater relative eye size than two northern drainages (Suwannee, North Florida). However, the results of our laboratory rearing experiment indicated that fish reared in clear water had slightly larger eyes compared to those in tea-stained water. While there are small effects of lighting environment on eye size, there are additional unknown genetic and environmental/ecological factors that influence adult eye size.