The microbial flora of the human vagina can affect the health of women, their fetuses, and newborns. Conventional cultivation methods fail to detect some fastidious vaginal bacteria, leading to an incomplete census. Recent advances in molecular biology have facilitated the detection and identification of bacteria without cultivation, and the advantages and limitations of this approach are described. Molecular studies of the vaginal flora have discovered many uncultivated bacterial species. For instance, several novel bacteria in the Clostridiales order are highly specific indicators of bacterial vaginosis, and bacteria related to Megasphaera, Leptotrichia, Atopobium, and Dialister species are commonly found in subjects with bacterial vaginosis. A more complete understanding of vaginal microbial populations resulting from the adoption of molecular tools may lead to better strategies to maintain healthy vaginal floras and will create opportunities to explore the role of novel bacteria in reproductive tract diseases.