In flies (Diptera), lineage-specific new genes (orphan genes), such as bicoid in the fruit fly Drosophila melanogaster and panish in the midge Chironomus riparius, have been found to establish head-to-tail polarity of the embryo. However, the embryonic axis determinants of most fly species have not been identified. In this study, I show that embryonic axis determinants of distantly related fly species have frequently evolved from old genes through the use of alternative transcription start or termination sites. In moth flies (Psychodidae), including the drain fly Clogmia albipunctata and the sand fly Lutzomyia longipalpis, a maternal transcript isoform of odd-paired (Zic) generated by alternative transcription initiation is localized in the anterior pole of freshly laid eggs, and is necessary and sufficient for anterior development of the Clogmia embryo. In the Southern house mosquito Culex quinquefasciatus (Culicidae), a maternal transcript isoform of a previously uncharacterized gene, named cucoid, is generated by alternative transcription termination (alternative polyadenylation), localized in the anterior pole of the egg, and necessary for establishing embryo polarity. This function of cucoid is also conserved in the yellow fever mosquito Aedes aegypti (Culicidae), suggesting a conserved role of this gene in axis specification in culicine mosquitoes. In the malaria mosquito Anopheles gambiae (Culicidae), a maternal transcript isoform of pangolin generated by alternative polyadenylation functions as the axis determinant. I also found localized maternal pangolin transcripts in the anterior eggs of Nephrotoma suturalis, a basal-branching crane fly (Tipulidae), suggesting that pangolin functioned as ancestral axis determinant in flies. This also suggests that panish, the pangolin-related orphan axis determinant gene of Chironomus, inherited its function from pangolin. Finally, I provide preliminary evidence for a diverged axis specification mechanism in the black soldier fly Hermetia illucens (Stratiomyidae), which is closely related to species with bicoid. I found that, unlike in other flies, numerous maternal mRNAs of conserved genes are localized in the anterior pole of the Hermetia egg. Together, I conclude that alternative transcription played an important role in the evolution of new developmental gene functions and gene regulatory networks in fly embryos. Given that alternative transcription is a widespread phenomenon and can underlie the evolution of entirely new gene functions, it may play a much more important role in animal evolution than previously thought.