Significance Our high-resolution, multiscale micro-computed tomography scans of Drosophila provide the first 3D view, to our knowledge, of the in situ morphological changes that occur in a female insect’s reproductive tract (RT) during and after mating. By means of this holistic analysis, we determined how the postmating reproductive events of ovulation, egg movement, sperm storage, sperm release, and fertilization are coordinated. We observed and quantified phenomena not detected in prior studies of dissected materials. These phenomena included coordinated looping and unlooping of the uterus and oviducts, and a potential mating-induced trauma in the female RT. This new perspective allowed us to revisit experimentally, and newly define, the effects of male- and female-secreted molecules on female postmating physiology.