Biogeographic barriers are thought to be important in initiating speciation through geographic isolation, but they rarely indiscriminately and completely reduce gene flow across the entire community. Understanding which species’ attributes regulate a barrier could help elucidate how speciation is initiated and isolation maintained. Here, we investigated the association of behavioral isolation on population differentiation in Northern Cardinals (Cardinalis cardinalis) distributed across the Cochise Filter Barrier, a region of transitional habitat which separates the Sonoran and Chihuahuan deserts of North America. Using genome-wide markers, we modeled demographic history by fitting the data to isolation and isolation-with-migration models. The best-fit model indicated that desert populations diverged in the Pleistocene with low, historic, and asymmetric gene flow across the barrier. We then tested behavioral isolation using reciprocal call-broadcast experiments to compare song recognition between deserts, controlling for song dialect changes within deserts. We found that male Northern Cardinals in both deserts were most aggressive to local songs and failed to recognize across-barrier songs. A correlation of genomic differentiation despite historic introgression and strong song discrimination is consistent with a model where speciation is initiated across a barrier and maintained by behavioral isolation.