Introduction and hypothesis <p>The relationship of adjacent pelvic microbiomes in women has not been well characterized. This study aimed to compare the microbiomes of catheterized urine, vagina, and perineum from well-characterized women undergoing urogynecologic surgery.</p> Methods <p>Clinical data was extracted from the electronic medical record. Participants contributed three samples (catheterized urine, vaginal swab, perineal swab) that were subjected to DNA sequencing following amplification of hyper-variable region 4 (V4) of the bacterial 16S rRNA gene. The sequences were processed, annotated, and decontaminated. Only participants with sequence-positive samples from all three samples were included in the analytic cohort. Alpha (within-sample) and beta (between-sample) diversity indices were computed, and the microbiome compositions within each participant and across all participants were compared.</p> Results <p>The mean age of the 104 participants was 60.5 years old (range 35–89) with a mean BMI of 28.2 kg/m²; most (89%) were White. <i>Lactobacillus</i> was the most common genus detected, with a mean relative abundance of 64.8% in the bladder, 67.4% in the vagina, and 42.6% in the perineum. Across these niches, alpha diversity differed significantly, whereas beta diversity was similar. Within an individual, the three microbiomes were very similar to each other, with the bladder and vagina considerably more similar to each other than to the perineum. Taxonomic diversity tended to increase with age and was highest in overweight participants.</p> Conclusion <p>The microbiomes of the bladder, vagina, and perineum of women are very similar to each other.</p>