This longitudinal study investigated neovaginal microbiota assembly in MRKH syndrome patients following laparoscopic peritoneal vaginoplasty. Early microbiota showed stochastic characteristics with *Enterococcus faecalis* and genital Mycoplasmas blooming. The pre-surgery dimple and fecal microbiota were primary species pools. At 6–12 months, the neovagina resembled a normal vagina but with a BV-like structure. At 2–4 years, it resembled the pre-surgery dimple microbiota. *Lactobacillus crispatus* colonization primarily originated from the dimple, suggesting avenues for promoting its colonization to improve neovaginal health.
