The members of the evolutionarily conserved Hox-gene complex, termed Hox genes, are required for specifying segmental identity during embryogenesis in various animal phyla. The Hox3 genes of winged insects have lost this ancestral function and are required for the development of extraembryonic epithelia, which do not contribute to any larval structure. Higher flies (Cyclorrhapha) such as Drosophila melanogaster contain Hox3 genes of two types, the zerknüllt type and the bicoid type. The zerknüllt gene is expressed zygotically on the dorsal side of the embryo and is required for establishing extraembryonic tissue. Its sister gene bicoid is expressed maternally and the transcripts are localized at the anterior pole of the mature egg. BICOID protein, which emerges from this localized source during early development, is required for embryonic patterning. All known direct bicoid homologues are confined to Cyclorrhaphan flies. Here, we describe Hox3 genes of the non-Cyclorrhaphan flies Empis livida (Empididae), Haematopota pluvialis (Tabanidae), and Clogmia albipunctata (Psychodidae). The gene sequences are more similar to zerknüllt homologues than to bicoid homologues, but they share expression characteristics of both genes. We propose that an ancestral Hox3 gene had been duplicated in the stem lineage of Cyclorrhaphan flies. During evolution, one of the gene copies lost maternal expression and evolved as zerknüllt, whereas the second copy lost zygotic expression and evolved as bicoid. Our finding correlates well with a partial reduction of zerknüllt-dependent extraembryonic tissue during Dipteran evolution.