Introduction: To understand the role of the urinary microbiome in disease states and interpret non-culture-based diagnostic urine testing of midstream urine specimens, we must have a better understanding of the urinary microbiome in asymptomatic healthy individuals. We examined the impact of gender, age, and menopausal status on the healthy human urinary microbiome in asymptomatic control subjects enrolled in the multi-institution National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Multidisciplinary Approach to the Study of Chronic Pelvic Pain Network (MAPP) study. Methods: Asymptomatic healthy controls, recruited to be age- and sex-matched to patients in the Trans-MAPP Epidemiology and Phenotyping Study, provided midstream urine collection for polymerase chain reaction (PCR)-electrospray ionization mass spectrometry identification of urinary microbiota. The microbiomes of male and female participants were described and analyzed for differences in composition and diversity at the species and genus level by sex, age, and, in females, by menopausal status. Results: Sixty-six total species were detected with a mean of 1.2 species (standard deviation [SD] 1.1) per male (n=97; mean age=43) and 2.3 (SD 1.3) per female (n=110, mean age=38) in asymptomatic healthy controls. Species and genera diversity analyses showed significantly greater richness and diversity in females. With regard to species, Bifidobacterium subtile, Lactobacillus crispatus, and Lactobacillus johnsonii were more predominant in females. The genera Bifidobacterium, Staphylococcus, Lactobacillus, and Corynebacterium were more predominant in females, while for males the most prevalent organisms included Staphylococcus and Propionibacterium; only Propionibacterium approached a significant difference between genders. No significant difference in the presence and/or diversity of micro-organisms with menopausal status could be observed. Sex-specific age trends, particularly diversity, were larger for females than males. Conclusions: These results suggest the urinary microbiome of healthy asymptomatic subjects differed between genders and age in females, but not menopausal status. Gender differences may be attributable to the detection of urethral/vaginal organisms in females and prostate organisms in males. These findings will better allow us to interpret the results of microbiome reports in the midstream urine specimens of patients with urinary symptoms.