ABSTRACT The human placenta was thought to be sterile in healthy pregnancies which has been challenged by the development of DNA sequence-based techniques, although it is still open to controversy. Nonetheless, little is known whether different parts of fetal appurtenances contain district microbiome profiles. Here, DNA 16S rRNA sequencing was performed of the amniotic fluid cells (AC), amnion membrane (AM), the placenta of fetal surface (remove the amniotic membrane, PL), maternal blood (MB), and umbilical cord blood (UCB) at V3-V4 hypervariable region from participants with cesarean delivery. Then sequence raw data were followed by taxonomic classification at 97% similarity and diversity analysis at the genus level. The differences and associations among the five tissues were analyzed. At the phylum composition level, the most abundant microorganisms were Proteobacteria in all five tissues, and followed by Firmicutes in AC, AM, and MB groups, Actinobacteria in UCB and Bacteroidetes in PL, respectively. As the maternal-fetal barrier, PL and AM had the lower OUT number and weaker co-occurrence network compared with the other three tissues. At the beta diversity clustering level, the microbiota constituents in the MB and UCB were highly similar; the microbiota profiles of PL and AM were also remarkably alike; AC was immensely different from those two clusters. Therefore, the five tissues were distinctly separated into three clusters. Our study reveals that different pregnancy-related anatomical sites harbor unique microbial compositions and show different degrees of correlation with other tissues.