We tested the hypothesis that the uterus of virgin heifers and pregnant cows possessed a resident microbiome. The endometrium of 10 virgin heifers in estrus and the amniotic fluid, placentome, intercotyleonary placenta, cervical lumen, and external cervix surface (control) of 5 pregnant cows were sampled using sterile surgical tools and aseptic surgical techniques. DNA was extracted, and the V4 hypervariable region of the 16S rRNA gene was amplified by PCR with barcode-indexed primers (U515F/806R), and sequenced. Operational taxonomic units were generated from the sequences, and taxonomy was assigned. The effect of tissue on the microbiome within the pregnant uterus was tested using univariate (mixed model) and multivariate (permutational multivariate ANOVA) procedures. Amplicons were generated in all samples supporting the contention that the uterus of virgin heifers and pregnant cows contained a microbiome. On average 53, 199, 380, 382, 525, and 13589 reads annotated as 16, 35, 43, 63, 48, and 176 OTUs in the placentome, virgin endometrium, amniotic fluid, cervical lumen, intercotyledonary placenta, and external surface of the cervix, respectively, were generated. The 3 most abundant phyla in the uteri of the virgin heifers and pregnant cows were Firmicutes, Bacteroidetes, and Proteobacteria and they accounted for approximately 40%, 35%, and 10% of the sequences, respectively. Phyla abundance was similar between the tissues of the pregnant uterus. Principal component analysis, one-way PERMANOVA analysis of the Bray-Curtis similarity index, and mixed model analysis of the Shannon diversity index and Chao1 index demonstrated that the microbiome of the control tissue was significantly different from the amniotic fluid, intercotyledonary placenta, and placentome tissues. Bacteria associated with postpartum uterine disease i.e., Trueperella spp., Fusobacteria spp., and Prevotella spp. were also present in the uterus of virgin heifers and of pregnant cows. The presence of 16S rRNA sequence reads in the samples from the current study suggests that the uterine microbiome is established by the time a female reaches reproductive maturity and that pregnancies are established and maintained in the presence of a uterine microbiome.