The flight ability of insects has coevolved with the development of organs necessary for takeoff from the ground, and to generate and modulate lift while flying in complex environments. Flight orientation to the appropriate food source and mating partner depends on the perception and integration of multiple chemical signals. We used a wind tunnel-based assay to investigate the natural and molecular evolution of free-flight odor-tracking behavior in Drosophila. First, the comparison of female and male flies of several populations and species revealed substantial sex-related, inter- and intra-specific variations for distinct flight features. In these flies, we compared the molecular structure of desat1, a fast-evolving gene involved in multiple aspects of pheromonal communication in Drosophila. We manipulated desat1 regulation and found that both neural and non-neural tissues influence distinct flight features. Together, our data suggest that desat1 is involved in the evolution of free-flight odor-tracking behaviors in Drosophila.