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ABSTRACT Sexual traits that promote species recognition are important drivers of reproductive isolation, especially among closely related species. Identifying neural processes that shape species differences in recognition is crucial for understanding the causal mechanisms of reproductive isolation. Temporal patterns are salient features of sexual signals that are widely used in species recognition by several taxa, including anurans. Recent advances in our understanding of temporal processing by the anuran auditory system provide an opportunity to investigate the neural basis of species-specific recognition. The anuran inferior colliculus consists of neurons that are selective for temporal features of calls. Of potential relevance are auditory neurons known as interval-counting neurons (ICNs) that are often selective for the pulse rate of conspecific advertisement calls. Here, we tested the hypothesis that ICNs mediate acoustic species recognition by exploiting the known differences in temporal selectivity in two cryptic species of gray treefrog (Hyla chrysoscelis and Hyla versicolor). We examined the extent to which the threshold number of pulses required to elicit behavioral responses from females and neural responses from ICNs was similar within each species but potentially different between the two species. In support of our hypothesis, we found that a species difference in behavioral pulse number thresholds closely matched the species difference in neural pulse number thresholds. However, this relationship held only for ICNs that exhibited band-pass tuning for conspecific pulse rates. Together, these findings suggest that differences in temporal processing of a subset of ICNs provide a mechanistic explanation for reproductive isolation between two cryptic treefrog species.