The South American fruit fly Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) cryptic species complex is presently one of the most studied pest models in terms of speciation and population mating compatibility. The improvement of pest-control techniques has strongly relied on successful implementation of laboratory strains into wild populations. Pheromone communication plays an important role in the mating process in the South American fruit fly. Therefore, the main goal of the present study was to investigate the pheromone composition of 7 different populations, originating from geographically distant locations in Brazil and Argentina. Fourteen volatile compounds were identified in calling male emanations by GC×GC/TOF-MS and the data obtained were subsequently analyzed by multivariate statistics. The pheromone composition varied both quantitatively and qualitatively among the studied populations. These results will serve as the basis for further electrophysiological analyses.