The region-specific homeotic gene spalt (sal) acts in two separate domains in the head and tail region of the Drosophila melanogaster embryo. Based on comparative morphology, sal is likely to be involved in the establishment of the head during the evolution of invertebrates and thus, it should be conserved. We have analyzed the conservation of the segmentation genes Krüppel (Kr) and even-skipped (eve) in parallel with sal coding sequences in several Drosophila species that are evolutionarily separated by up to 60 million years. To our surprise, sal sequences appear to be conserved in the Sophophora subgenus of the Drosophila genus but not in the Drosophila subgenus. On the other hand, the segmentation and other homeotic genes are conserved in the Drosophila subgroup as well. Our data suggest that sal encodes an accessory function that evolved relatively late during Drosophila speciation rather than playing a fundamental evolutionary role similar to that of other homeotic genes.