Animals use acoustic signals to repel competitors and attract mates, and signal divergence among populations can promote reproductive isolation. Empidonax flycatchers are insectivorous passerine birds distributed across North and Central America that are conservative in plumage but often exhibit differences in song both between and within species. Four subspecies have been recognized within the Willow Flycatcher (Empidonax traillii) and previous analyses have revealed differences in song structure among a subset of these.  Using reciprocal playback experiments in the field, we tested for subspecific song discrimination among these four putative Willow Flycatcher subspecies. We found that two of the western subspecies (E. t. adastus and E. t. brewsteri) showed comparable aggressive responses to playbacks of their own song and those of each other and the eastern subspecies (E. t. traillii) while exhibiting significantly lower responses to songs of the southwestern subspecies (E. t. extimus). The eastern subspecies (E. t. traillii) likewise exhibited aggressive responses to its own song significantly greater than that to the southwestern subspecies (E. t. extimus) but variable responses to E. t. adastus and E. t. brewsteri. In contrast, the southwestern subspecies (E. t. extimus) responded more aggressively to its own song relative to all other subspecies. Our results indicate that behavioral responses reflect differences in song structure among subspecies; subspecies with similar song structure responded more strongly to subspecies with that structure and less strongly to songs most different in structure, while the subspecies with the most distinctive song responded less to songs of the other three. If male response to song reflects relative reproductive compatibility within and among subspecies, song may contribute to reproductive isolation between Willow Flycatcher subspecies.