Understanding the distribution patterns of closely related species is critical because the extent to which their ranges overlap determines the opportunity for competition and hybridization. In this study, we used museum records to determine the degree of overlap between two killifish species, Lucania goodei and L. parva, in Florida. While the broad geographic distributions and habitat characteristics are well-described, the degree of sympatry between the two species, the temporal stability of sympatric populations, and the abiotic and biotic conditions under which sympatric populations occur are unknown. Using the museum records, we identified sites where L. goodei and/or L. parva had been collected and classified sites as either L. goodei–allopatric, L. parva–allopatric, or sympatric. For sites that were sampled repeatedly over time, we determined the extent to which their status varied. Approximately 12–19% of sites where L. goodei was present were sympatric with L. parva at some point in time. However, many sympatric sites were not stable over time. Of the repeatedly sampled sites that were sympatric at some point in time, more than 50% varied between allopatric and sympatric status. Salinity also had large effects on distribution. Approximately two-thirds of sympatric sites were in fresh water, and the remaining one-third were in brackish water. As expected, the fish community varied between L. goodei, L. parva, and sympatric sites. The unique contribution of this study is that it provides the first estimate of the degree of overlap between L. goodei and L. parva and indicates which biotic and abiotic variables may account for this pattern.