Summary An approximately 220 bp fragment of the internal transcribed spacer 2 (ITS2) was screened as a diagnostic trait of Anastrepha suspensa (Loew) (Diptera: Tephritidae) and other pest fruit fly species in the genus Anastrepha. The majority (96%) of the sites in this fragment were invariant among the test species, but A. suspensa can be separated from other species by using 2 diagnostic characters. Similarly, A. ludens (Loew) and A. obliqua (Macquart) can be distinguished from other species based on 1 fixed character each. There is evidence of intraspecific ITS2 variation in 5 species tested, consistent with species complexes and incomplete homogenization through the process of concerted evolution.