Vaginal colonization with lactobacilli has been linked to the health of the lower urinary tract in women. There is growing evidence that the bladder has its microbiome related closely to the vagina. In this study, we compared the three common vaginal Lactobacillus species (L. jensenii, L. iners and L. crispatus) in vaginal and urine samples to identify factors that influence urinary detection and the quantity of Lactobacillus. We used quantitative real-time PCR (qPCR) assays to measure the concentration of Lactobacillus jensenii, L. iners and L. crispatus in paired vaginal swabs and clean-catch urine samples from pre-and post-menopausal women. We compared demographic variables and vaginal Lactobacillus quantity between women with vaginal detection of at least one of the three species, detection in both vagina and urine, or urine only. We performed Spearman correlation between vaginal and urinary quantities of each species. We used multivariable logistic regression models to determine predictors of detectable Lactobacillus species in both samples (vs. vagina only or urine only). Models were adjusted for variables selected a priori: age, BMI, condom use, and recent sexual activity. Ninety-three paired vaginal fluid, and urine samples were included in the final analysis. 44 (47%) had no detectable Lactobacillus species in their urine samples, and 49 (53%) had at least one of the three Lactobacillus species (L. jensenii, L. iners and L. crispatus) detected in urine. Most women were white (91.4%), with a mean age of 39.8 ±13.8 years. The two groups were similar in demographics, gynecologic history, sexual history, recent use of antibiotics or probiotics within 7 days of sample collection, Nugent scores, and urine-specific gravity. Among the three Lactobacillus species, L. jensenii was more commonly detected in urine than the other two. For all three species, detection in the urine sample alone was infrequent. The concentrations of all three species were higher in vaginal samples than in urine samples. For all three Lactobacillus spp., vaginal abundance was associated with the urinary abundance of the same species even after adjusting for the Nugent score. In Spearman correlation analysis, urinary and vaginal Lactobacillus concentrations were positively correlated within the same species, with the most significant correlation coefficient for L. jensenii (R = 0.43, p<0.0001). Vaginal quantities were positively correlated between the three species, as were urinary quantities to a lesser extent. There was no meaningful correlation between the urinary quantity of one Lactobacillus sp. and the vaginal quantity of another species. In summary, the vaginal quantity of Lactobacillus was the most significant predictor of concurrent detection of the same species in the bladder, confirming the close relationship between these environments. Strategies to promote vaginal Lactobacillus colonization may also bring urinary colonization and the health of the lower urinary tract.