Neovaginas are surgically constructed to correct uterovaginal agenesis in women with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. Here, in a longitudinal study, the authors characterize the dynamics of neovaginal microbiota assembly, showing resemblance to normal vaginal flora at 6-12 months post-surgery, albeit with a bacterial vaginosis-like structure, and further developed into a structure resembling that of the homeostatic pre-surgery dimple microbiota by 2-4 years post-surgery.