OBJECTIVE: To compare the endometrial and vaginal microbiome of women with and without chronic endometritis. STUDY DESIGN: A cohort study with 60 patients undergoing assisted reproductive treatment with their own or donated gametes was undertaken. Vaginal and endometrial samples were taken in the cycle prior to embryo transfer. The endometrial and vaginal microbiome was analysed by mass sequencing of the V3V4 region of 16S rRNA gene. Bioinformatics analysis was performed using QIIME2 and MicrobiomeAnalyst packages. Alpha diversity, beta diversity and taxonomic characterization were compared between samples that tested positive and negative for chronic endometritis on CD138 immunohistochemistry. RESULTS: Different bacterial communities were detected when vaginal and endometrial samples were analysed in patients with and without endometritis diagnosed using CD138 immunohistochemistry. In patients with endometritis, a higher alpha-diversity index was found in vaginal samples (p = 0.15 for the Shannon index) and significant differences were found in endometrial samples (p = 0.01 for the Shannon index). In the beta-diversity analysis, no significant differences were observed between the groups with and without endometritis. Vaginal and endometrial samples from women with endometritis showed a microbiome pattern that was not dominated by Lactobacillus spp. Relative abundance analysis identified Ralstonia and Gardnerella spp. in endometrial samples, and Streptoccoccus and Ureaplasma spp. in vaginal samples of patients diagnosed with chronic endometritis on CD138 immunohistochemistry. When comparing endometrial and vaginal samples diagnosed with endometritis on CD138 immunohistochemistry, both alpha diversity (p = 0.06 for the Shannon index and p = 0.08 for the Simpson index) and beta diversity (p < 0.001) showed significant differences. Lactobacillus spp. (p = 3.76E-4), Ralstonia spp. (p = 8.19E-4), Delftia spp. (p = 0.004) and Anaerobacillus spp. (p = 0.004) were identified in these sample groups. CONCLUSION: These results demonstrate the existence of a characteristic vaginal and endometrial microbiota in patients with chronic endometritis. Different genera and species were identified in patients with and without chronic endometritis depending on whether the sample was endometrial or vaginal. There is a clear relationship between changes in the vaginal microbiome and chronic endometritis. The microbiota is a continuum throughout the female reproductive tract, so study of the vaginal microbiota could be useful for the diagnosis of diseases of the upper reproductive tract, such as chronic endometritis.