© 2017 The Author(s) Cell migration is indispensable to morphogenesis and homeostasis. Live imaging allows mechanistic insights, but long-term observation can alter normal biology, and tools to track movements in vivo without perturbation are lacking. We develop here a tool called M-TRAIL (matrix-labeling technique for real-time and inferred location), which reveals migration histories in fixed tissues. Using clones that overexpress GFP-tagged extracellular matrix (ECM) components, motility trajectories are mapped based on durable traces deposited onto basement membrane. We applied M-TRAIL to Drosophila follicle rotation, comparing in vivo and ex vivo migratory dynamics. The rate, trajectory, and cessation of rotation in wild-type (WT) follicles measured in vivo and ex vivo were identical, as was rotation failure in fat2 mutants. However, follicles carrying intracellularly truncated Fat2, previously reported to lack rotation ex vivo, in fact rotate in vivo at a reduced speed, thus revalidating the hypothesis that rotation is required for tissue elongation. The M-TRAIL approach could be applied to track and quantitate in vivo cell motility in other tissues and organisms. Chen et al. describe M-TRAIL, a new ECM-based tool for tracking cell migration in living tissues, and demonstrate its utility in resolving conflicting models linking migration and morphogenesis in Drosophila egg chambers.