Studies in recent years indicate that microbial communities residing in the reproductive tract are crucial for shaping mammals’ health and reproductive outcomes. Following parturition, female mammals often exhibit uterine bacterial contamination due to the open cervix allowing microbes from the vaginal microbiota or the environment to enter. This contamination may be associated with postpartum uterine inflammatory diseases, which are more prevalent among primiparous than multiparous individuals, as reported in dairy cows and women. However, investigations into the spatio-temporal microbial transitions in the reproductive tract of primigravid heifers and women remain limited. Our objective was to describe and compare the community compositions in the vagina at late gestation and in the vagina and uterus at early postpartum, in first-pregnancy heifers. Three swab samples were collected from 33 first-pregnancy Holstein-Friesian heifers: vaginal samples at gestation day 258±4, and vaginal and uterine samples at postpartum day 7±2. Each sample underwent 16S rRNA V4 region metagenetic analysis via Illumina MiSeq, with bioinformatics following Mothur MiSeq SOP. Results revealed distinct shifts in microbial composition between the prepartum vagina, postpartum vagina, and postpartum uterus. The late gestation vaginal and early postpartum uterine microbial profiles were the most distinct, with postpartum vaginal samples serving as intermediaries. The postpartum uterus exhibited lower richness and diversity compared to the prepartum and postpartum vagina. Specific microbial markers were associated with anatomical and temporal transitions, indicating a dynamic microbial landscape associated with parturition. The observed differences in phyla and genera across reproductive sites highlight the complexity of these ecosystems, expending avenues for future research in reproductive biology, microbial ecology, and reproductive tract diseases.