More than 1.3 million US adults identify as transgender or gender diverse (TGD), meaning their sex assigned at birth does not align with their current gender identity. Transmasculine individuals (TM), who were assigned female at birth but experience male or masculine gender identity, may utilize gender-affirming testosterone therapy to more closely align their gender presentation with their internal sense of gender. Some individuals report adverse genital symptoms while using testosterone therapy, such as dryness, non-menstrual bleeding, pain during sex, and vaginal atrophy. In reproductive-aged cis females (rCF), the vaginal microbiota is a key determinant of reproductive and sexual health outcomes, with Lactobacillus-dominant communities being considered optimal. Conversely, more even, diverse microbiota in rCF have been associated with non-optimal outcomes including pre-term birth, increased HIV and other STI acquisition, and bacterial vaginosis (BV). The few studies that have characterized the vaginal microbiota in TM have observed that many individuals exhibit a Lactobacillus-deficient microbiota, though study design did not allow for associations to be determined with symptomology and local immune status. We therefore launched the TransBiota study to characterize the composition and structure of the TM vaginal microbiota, soluble mediators of local inflammation (SMI), and self-reported symptoms over three weekly samples. We found that less than 10% of sampled TM possessed Lactobacillus-dominant microbiota, while the majority possessed more diverse, even microbiota, somewhat akin to the vaginal microbiota of postmenopausal women. We identified 11 biologically relevant clusters of vaginal microbiota in TM (tmCSTs); unexpectedly, Lactobacillus-dominant tmCSTs were associated with increased reports of abnormal or unpleasant odor and elevated IL-1α concentrations. Comparative genomic analyses highlighted the similarities between TM and rCF Lactobacillus crispatus, Streptococcus anginosus, and Prevotella bivia genomes, while revealing the differences between Prevotella timonensis and Lactobacillus iners in TM versus rCF. Together, these findings indicate that perhaps Lactobacillus dominance is no longer an optimal state for transmasculine individuals during gender-affirming testosterone therapy and highlight the need to adapt clinical care guidelines for transmasculine individuals presenting for gynecological care during gender affirming testosterone treatment.