The successful hybridization of cosmid clones from Drosophila melanogaster (Sophophora subgenus) to the salivary gland chromosomes of other species as distantly related as those in the Drosophila subgenus attests their great potential for unravelling genome evolution. We have carried out, using 28 cosmids and 13 gene clones, a study of the organization of the D. melanogaster 95A-96A chromosomal region in three Drosophila subgenus species: D. repleta, D. buzzattii and D. virilis. These clones were first used to built an accurate map of this 1.6 Mb region of D. melanogaster chromosome 3R (Muller's element E). Then, they were hybridized and mapped to the homologous chromosome 2 of the other three distantly related species. The studied region is disseminated over 13 different sites of chromosome 2 in the Drosophila subgenus species, which implies a minimum of 12 inversion breakpoints fixed between the two subgenera. Extrapolation to the entire chromosome gives 90 fixed inversions. The D. melanogaster Pp1-96A-Acr96Aa segment conserved in D. repleta and D. buzzatii is longer than previously thought and is also conserved in D. virilis. In addition, three other D. melanogaster segments conserved in the three Drosophila subgenus species were found. Finally, our data indicate significant statistical differences in the evolution rate of Muller's element E among lineages, a result that agrees well with the previous cytogenetic data.