Freshwater fishes exhibit a high diversity of colors and color patterns. In particular, fishes of the genus Etheostoma have colors perceived by humans as red, orange, yellow, green, and blue in a variety of combinations. We used reflectance spectrophotometry to evaluate color diversity quantitatively among four species of Etheostoma: E. baileyi, E. coosae, E. zonale, and E. zonistium. Three spectral types were identified, corresponding to colors perceived by humans as ‘blue’ or ‘green,’ ‘red’ or ‘orange,’ and ‘yellow.’ Species differences in the peak wavelengths of the ‘blue’ and ‘green’ spectra were observed, and these complex spectra also differed in reflectance in the long-wavelength portion of the spectrum. Species also differed in the midpoint reflectance of ‘red’ and ‘orange’ spectra. By presenting preliminary descriptions of spectral properties of breeding males, we provide a framework for further investigations into the evolution of breeding coloration and visual ecology in darters.