Mating compatibility among different populations of the South American fruit fly Anastrepha fraterculus (Wiedemann) was assessed through mating tests in pairwise combinations. Screened cages, inside a greenhouse, containing Citrus limon (L.) trees were used. Mating compatibility was determined using the index of sexual isolation. Most of the populations were noncompatible with each other and thus sexually isolated. Of these, Tucumán (Argentina) and Piracicaba (Brazil) populations showed a lower degree of isolation, whereas the other tested combinations were highly isolated. Full mating compatibility was detected only between two Argentinean (Concordia and Tucumán) and two Peruvian populations (La Molina and Piura La Molina). Flies were sexually active at different times: Tucumán, Concordia, and Piracicaba populations presented an early morning peak, La Molina and Piura La Molina were active around midday, and Ibague (Colombia) were active late in the afternoon. Manipulation of light phase conditions to match the times of maximum sexual activity did not increase the compatibility between La Molina and Tucumán. Based on these behavioral results, which confirm morphometric, genetic, and other evidence, the taxonomic revision of this cryptic species complex is warranted. One practical implication is that colonies of this pest to be used in any sterile insect technique approach should be derived from the target population or from a compatible population. Regional efforts should be initiated to determine the distribution of each subgroup and their relationship with each other in terms of compatibility.