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Objective: The aim of this study was to evaluate the differences between the vaginal microbiome of reproductive-aged women with overweight and obesity (Ow/Ob) compared with healthy weight (HW)., Methods: In this case-control study, a cohort of 367 nonpregnant women (18 to 40 years) with Ow/Ob (BMI ≥25 kg/m 2 ) was case-matched with 367 women with HW (BMI 18.0 to 24.9 kg/m 2 ). The study was a secondary analysis of 16S rRNA vaginal microbiome surveys through the Vaginal Human Microbiome Study (VaHMP). Groups were matched on age, race/ethnicity, income, and nulliparity status., Results: Mean age and BMI of Ow/Ob and HW groups were 26.8 versus 26.7 years and 37.0 versus 22.1 kg/m 2 , respectively. The overall vaginal microbiome composition differed between groups (PERMANOVA, p = 0.035). Women with Ow/Ob had higher alpha diversity compared with women with HW (Wilcoxon test, Shannon index p = 0.025; inverse Simpson index p = 0.026). Lactobacillus dominance (≥30% proportional abundance) was observed in a greater proportion of women with HW (48.7%) compared with Ow/Ob (40.1%; p = 0.026)., Conclusions: The vaginal microbiome differs in reproductive-aged women with Ow/Ob compared with women with HW, with increased alpha diversity and decreased predominance of Lactobacillus. Observed differences in the vaginal microbiome may partially explain differences in preterm birth and bacterial vaginosis risk between these populations., (© 2021 The Obesity Society.)