Abstract Flying insects have invaded all the aerial space on Earth and this astonishing radiation could not have been possible without a remarkable morphological diversification of their flight appendages. Here, we show that characteristic spatial expression profiles and levels of the Hox genes Antennapedia ( Antp ) and Ultrabithorax ( Ubx ) underlie the formation of two different flight organs in the fruit fly Drosophila melanogaster . We further demonstrate that flight appendage morphology is dependent on specific Hox doses. Interestingly, we find that wing morphology from evolutionary distant four-winged insect species is also associated with a differential expression of Antp and Ubx . We propose that variation in the spatial expression profile and dosage of Hox proteins is a major determinant of flight appendage diversification in Drosophila and possibly in other insect species during evolution.