In many species, frequency-dependent sexual selection (FdSs) influences fitness. We explored the potential for the presence of FdSs in humans by studying the relationship between eye color frequency and rankings of a person's attractiveness. Standardized photographs were sorted into sets of six faces with similar characteristics. Each image was edited to have one of three eye colors. Subjects then ranked the attractiveness of face sets in which the ratios of two eye colors were altered. Relationships between the eye color frequency and their attractiveness ranking indicate frequency-dependent preference by the subjects. Trends towards negative FdSs were present in males and females. Significant results for white females and for Asian-American males exhibiting negative FdSs were found. We did not find significant results for imprinting (based on opposite-sex parent eye color) in males nor in females. We conclude that negative FdSs affects human attractiveness in some populations.