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The vaginal microbiome is an emerging concern in prenatal health. Because the sampling process of vaginal microbiota may pose potential risks for pregnant women, the choice of sampling site should be carefully considered. However, whether the microbial diversity is different across various sampling sites has been controversial. In the present study, three repeated swabs were collected at the cervix (C), posterior fornix (P), and vaginal canal (V) from 34 Chinese women during different pregnancy stages, and vaginal species were determined using the Illumina sequencing of 16S rRNA tag sequences. The identified microbiomes were classified into four community state types (CSTs): CST I (dominated by L. crispatus), CST II (dominated by L. gasseri), CST III (dominated by L. iners), and CST IV-A (characterized by a low abundance of Lactobacillus, but with proportions of various species previously shown to be associated with bacterial vaginosis). All individuals had consistent CST at the three sampling sites regardless of pregnancy stage and CST group. In addition, there was little heterogeneity across community structures within each individual, as determined by LEfSe, indicating high vaginal microbiome homogeneity at the three sampling sites. The present study also revealed different beta diversity during pregnancy stages. The vaginal microbiome variation among women during trimester T1 (9 ± 2.6 weeks) is larger than that of non-pregnant women and women from other trimesters, as demonstrated by the UniFrac distance (P < 0.05). In particular, the present study is the first one that demonstrates the notably difference of vaginal microbiome of postpartum women compare to women in gestation. These results will be useful for future studies of the vaginal microbiota during pregnancy.

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