The prevalence of bacterial vaginosis among lesbians is high. Weassessed whether unique Lactobacillus strains were shared by female sex partners. Cultures of vaginal and rectal specimens for detection of Lactobacillus organisms were performed for women who reported having had sex with women. Lactobacilli were identified on the basis of DNA homology and were typed and fingerprinted by repetitive element sequence-based polymerase chain reaction (rep-PCR). Of 237 women, Lactobacillus crispatus was detected in 98% and Lactobacillus gasseri in 21%. Detection of L. gasseri was associated with recent receptive digital-vaginal sex (P = .04) and increased bacterial vaginosis risk (odds ratio, 4.2; 95% confidence interval, 1.4-13.4). Of 31 couples monogamous for >3 months, rep-PCR fingerprints were identical in both members in 23 (77%). © 2009 by the Infectious Diseases Society of America. All rights reserved.