The doublesex (dsx) gene of several Anastrepha species was isolated and characterised. Its molecular organisation was found to be the same in all the species examined. This gene is composed of four exons: Exons 1 and 2 are common to both sexes, exon 3 is female specific, and exon 4 is male specific. It codes for both the female DsxF and male DsxM proteins, corresponding to the sex-specific splicing product of its primary transcript; male-specific splicing is the default mode. A comparison of the Dsx proteins of different Anastrepha species with those of other insects showed them to be very similar. Molecular evolutionary analysis (both at the nucleotide and amino acid levels) of dsx in different insects revealed a topology in good agreement with their owners' taxonomic relationships. The great majority of the nucleotide changes detected in the dsx gene of the analysed species were significantly synonymous, evidence that strong purifying selection has acted on dsx so that the functional structure of the Dsx proteins is preserved. However, the common region of DsxF and DsxM proteins appeared to be the main target for selection acting upon the long-term evolution of gene dsx.