Geographically isolated populations of birds often differ in song. Because birds often choose mates on the basis of their song, song differentiation between isolated populations constitutes a behavioral barrier to reproduction. If this barrier is judged to be sufficiently strong, then isolated populations with divergent songs may merit classification as distinct species under the biological species concept. We used a dataset of 72 pairs of related but allopatric Neotropical passerines (“taxon pairs”) to compare 2 methods for measuring song divergence between isolated populations: statistical analysis of 7 acoustic traits measured from spectrograms, and field playback experiments that “ask the birds themselves” if they perceive foreign song as conspecific or not. We report 4 main findings: (1) Behavioral discrimination (defined as failure to approach the speaker in response to allopatric song) is nonlinearly related to divergence in acoustic traits; discrimination is variable at low to moderate levels of acoustic divergence, but nearly uniformly high at high levels. (2) The same nonlinear relationship held for both song learners (oscines) and nonlearners (suboscines). (3) Song discrimination is not greater in taxon pairs ranked as species compared to taxon pairs ranked as subspecies. (4) Behavioral responses to allopatric song are symmetric within a taxon pair. We conclude (1) that playback experiments provide a stronger measure of species recognition relevant to premating reproductive isolation than do acoustic trait analyses, at least when divergence in acoustic traits is low to moderate; and (2) that playback experiments are useful for defining species limits and can help address the latitudinal gradient in taxonomy, which arises because species are defined more broadly in the tropics than in the temperate zone. To this end, we suggest that 21 Neotropical taxon pairs that are currently ranked as subspecies, but that show strong behavioral discrimination in response to allopatric song, merit classification as distinct biological species.