We examined the urethral microbiota, determined if it differs from the bladder urinary microbiota, and assessed if its composition differs based on patient demographic factors and presence of lower urinary tract symptoms. Patients presenting to our urogynecology clinic were enrolled in the study. Demographic information and responses to the Pelvic Floor Distress Inventory questionnaire were collected. All participants provided midstream voided urine, periurethral swab, transurethral swab and catheterized urine samples, which were analyzed by Expanded Quantitative Urine Culture and MALDI-TOF mass spectrometry. Bray-Curtis dissimilarity analysis assessed diversity between sample types for each participant. Kruskal-Wallis, chi-square, McNemar, Wilcoxon signed rank and Fisher's exact tests tested for significance. A total of 49 patients participated in the study. Bladder microbiota were dissimilar to urethral, periurethral and voided urine microbiota (p <0.0001). Urethral and periurethral microbiota were similar (p >0.05), but the urethral microbiota were dissimilar to voided urine microbiota (p=0.001) while the periurethral microbiota were not (p >0.05). Women less than 55 years old were more likely to be sexually active, premenopausal and Hispanic compared to women 55 years old or older. Women in the younger cohort had Lactobacillus and Gardnerella cultured from urethral samples more frequently and more abundantly than women in the older cohort. There was no significant association between lower urinary tract symptoms and the frequency or abundance of urethral bacteria species. Niches of microbiota along the female lower urinary tract may be influenced by age, menopausal status and sexual activity. More research is needed to determine the function and clinical significance of the urethral microbiome.