Phylogenetic relationships among Andean-Ecuadorian and other Neotropical populations of Anastrepha fraterculus and related species have been studied using two regions of mtDNA : 405 base pairs within Cytochrome Oxidase I (COI) and 224 base pairs within Cytochrome Oxidase II (COII). Phylogenetic relationships were inferred using Maximun Parsimony (MP) method and haplotype networks. Andean-Ecuadorian populations of A. fraterculus are monomorphic at the COI locus and fall within a clade of South-American lowland populations of A. fraterculus. They appear to be unrelated with populations of northern Andes of Colombia and Venezuela also assigned to A. fraterculus, meaning that this species, as currently circumscribed, is not monophyletic and is composed of different biological entities that are little differentiated morphologically. At the COII locus, Andean-Ecuadorian populations of A. fraterculus show a major haplotype with a few variants, and form a clade with the lowland populations of southern Brazil an Argentina, but are clearly differentiated from them. Andean-Ecuadorian populations of Anastrepha fraterculus appear to be homogeneous with respect to their mitochondrial genome and thus their identity as members of a single gene pool is confirmed by these results.