<p>Different populations of a species may overlap geographically with closely related taxa, increasing the risk of maladaptive hybridization. In such contact zones, male advertisement calls and corresponding female preferences can diverge between sympatric and allopatric populations, contributing to reproductive isolation. The neural basis of these population-level differences in communication, however, remains poorly understood. In the Upland chorus frog (<i>Pseudacris feriarum</i>), sympatric populations produce advertisement calls with faster pulse rates and/or more pulses per call than allopatric populations. To examine potential neural correlates of this divergence, we recorded from long-interval neurons (LINs) in the auditory midbrain using in vivo extracellular methods and compared their temporal selectivity across populations. LINs from allopatric populations responded to a wider range of pulse rates, extending to faster rates than LINs from sympatric populations, which responded primarily to slower pulse rates characteristic of heterospecific calls. These findings demonstrate population-level differences in LIN temporal tuning and reveal a mismatch between pulse rate selectivity and the temporal properties of sympatric advertisement calls. Our results reveal that LINs are a target of reinforcing selection, and provide initial evidence toward understanding how auditory processing differs across populations experiencing different selective pressures.</p>