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Geographic variation in courtship behavior can affect reproductive success of divergent phenotypes via mate choice. Over time, this can lead to reproductive isolation and ultimately to speciation. The Neotropical red-eyed treefrog ( Agalychnis callidryas) exhibits high levels of phenotypic variation among populations in Costa Rica and Panama, including differences in color pattern, body size, and skin peptides. To test the extent of behavioral premating isolation among differentiated populations, we quantified male advertisement calls from six sites and female responses to male stimuli (acoustic and visual signals) from four sites. Our results show that both male advertisement calls and female behavior vary among populations: Discriminant function analyses can predict the population of origin for 99.3% ± 0.7 of males based on male call (dominant frequency and bandwidth) and 76.1% ± 6.6 of females based on female response behavior (frequency and duration of visual displays). Further, female mate choice trials ( n = 69) showed that population divergence in male signals is coupled with female preference for local male stimuli. Combined, these results suggest that evolved differences among populations in male call properties and female response signals could have consequences for reproductive isolation. Finally, population variation in male and female behavior was not well explained by geographic or genetic distance, indicating a role for localized selection and/or drift. The interplay between male courtship and female responses may facilitate the evolution of local variants in courtship style, thus accelerating premating isolation via assortative mating. [ABSTRACT FROM AUTHOR]