It is not known whether the auditory hair cells of fishes possess a tonotopic organization in the saccule. To investigate this question, we exposed groups of six goldfish (Carassius auratus) to one of four tones (100, 800, 2000, and 4000 Hz) at 176 dB re 1 μPa for 48 h. The saccules of each fish were dissected and labeled with phalloidin in order to visualize hair cell bundles. The hair cell bundles were counted at 19 specific points in each saccule to determine the extent and location of hair cell damage. In addition to quantification of anatomical injury, hearing tests (using auditory evoked potentials) were performed on each fish immediately following sound exposure. The location of hair cell loss varied along the length of the saccule in a graded manner with the frequency of sound exposure, with lower and higher frequencies damaging the more caudal and rostral regions of the saccule, respectively. Similarly, fish exposed to lower frequency tones exhibited greater threshold shifts at lower frequencies, while high-frequency tone exposure led to hearing loss at higher frequencies. These data suggest that the frequency discrimination ability of goldfish is at least partially driven by peripheral tonotopy in the saccule.