Divergence in vocalizations can reduce gene flow by serving as a premating barrier during secondary contact between previously isolated populations. In primates, vocal divergence in long calls of separated populations has been documented, yet recognition of these differences by the respective populations has seldom been studied in the field. To investigate this issue, we studied populations of two subspecies of saddle‐back tamarins (Saguinus fuscicollis nigrifrons and S. f. lagonotus) that are separated by the Amazon River in Peru. We recorded long calls of each subspecies and detected significant differences between the populations in the number of notes per call, duration of calls, and shifts in starting frequency of notes over the length of calls. In addition, a population of S. f. nigrifrons responded more overtly in measures of approach to playback of long calls of its own subspecies compared to long calls of S. f. lagonotus. These results are consistent with the hypothesis that allopatric divergence of long calls might contribute to reproductive isolation of these subspecies of saddle‐back tamarins, which adds to growing evidence suggesting full species status for these taxa. Am. J. Primatol. 77:679–687, 2015. © 2015 Wiley Periodicals, Inc.