Evolutionary divergence between species is facilitated by ecological shifts, and divergence is particularly rapid when such shifts also promote assortative mating. Horseshoe bats are a diverse Old World family (Rhinolophidae) that have undergone a rapid radiation in the past 5 million years. These insectivorous bats use a predominantly pure-tone echolocation call matched to an auditory fovea (an over-representation of the pure-tone frequency in the cochlea and inferior colliculus) to detect the minute changes in echo amplitude and frequency generated when an insect flutters its wings. The emitted signal is the accentuated second harmonic of a series in which the fundamental and remaining harmonics are filtered out. Here we show that three distinct, sympatric size morphs of the large-eared horseshoe bat (Rhinolophus philippinensis) echolocate at different harmonics of the same fundamental frequency. These morphs have undergone recent genetic divergence, and this process has occurred in parallel more than once. We suggest that switching harmonics creates a discontinuity in the bats' perception of available prey that can initiate disruptive selection. Moreover, because call frequency in horseshoe bats has a dual function in resource acquisition and communication, ecological selection on frequency might lead to assortative mating and ultimately reproductive isolation and speciation, regardless of external barriers to gene flow.