Mate recognition systems, both the signals produced and the sensory mechanisms to receive them, often diverge to result in pre-mating reproductive isolation between closely related species. Comparative and experimental studies of anurans have contributed to our understanding of this process. By comparing populations of related species in allopatry, sympatry, and syntopy, it is possible to identify the environmental factors associated with divergence in mate recognition systems, including interactions with related species. Many pairs of species meet along the Fall Zone in the Southeast, but few have been well researched. My study compared the two species of cricket frogs, Acris crepitans and A. gryllus, at 36 sites in North Carolina. I assessed the acoustic and morphological traits used to identify the species, determined the extent of their ranges and sympatry, and identified 4 syntopic sites in the upper Coastal Plain. The dominant frequency and call rate of male vocalizations varied widely and overlapped between the species. Body mass had the largest effect on these features. In contrast, the effects of seasonality and temperature were minor. Additional variation between sites could not be attributed to sympatry or syntopy, so there was no evidence of reproductive character displacement in dominant frequency or call rate. In playback experiments at a syntopic site, females of both species discriminated between conspecific and heterospecific signals on the basis of click structure, a fine-scale temporal feature, and had no preference for dominant frequency among conspecific signals. Reproductive isolation in Acris is promoted by divergence in the temporal structure of male signals. Only studies like this one, conducted on geographic scales appropriate for comparisons of local populations, can identify patterns of geographic variation in signals that contribute to reproductive isolation.