We examined the utility for phylogenetic reconstruction of the second internal transcribed spacer (ITS2), lying between the nuclear 5.8S gene and the gene for large subunit ribosomal RNA, using sequences of Ceratitis, Bactrocera, Musca, and Drosophila. We aligned and analyzed 13 sequences from GenBank and 11 new sequences from diverse species of Drosophila. Derivation of the secondary structure of the ITS2, the RNA transcript folding pattern required for transcript processing into functional RNA units, revealed the facets of sequence conservation common to all the sequences, that then allowed alignment of all the genera. The resultant tree, though including only a sparse representation of the enormous Drosophila diversity, conforms generally with the consensus of all prior phylogenetic reconstructions, using eight other nuclear and mitochondrial gene regions; where species representation is greater, as in the melanogaster subgroup of the Sophophora subgenus representatives, it conforms exactly. The paradigm ITS2 secondary structure presented can now be used to assess the genus more thoroughly, since its base pairing pattern makes alignment of sequences obvious. In addition, it shows that these insects share the ITS2 secondary structure characteristics of the other major animal groups as well as the green line of eukaryote evolution. The relatively short (<400 bp) ITS2 region seems ideal for reconstructing evolutionary relationships at the levels of species, genera, and perhaps even higher.