AbstractBackgroundGardnerella vaginalisis a hallmark of vaginal dysbiosis, but is found in the microbiomes of women with and without vaginal symptoms.G. vaginalisencompasses diverse taxa differing in attributes that are potentially important for virulence, and there is evidence that ‘clades’ or ‘subgroups’ within the species are differentially associated with clinical outcomes. TheG. vaginalisspecies description was recently emended, and three new species within the genus were defined (leopoldii,swidsinskii,piotii). 16S rRNA sequences for the fourGardnerellaspecies are all >98.5% identical and no signature sequences differentiate them.ResultsWe demonstrated thatGardnerellaspecies can be resolved using partial chaperonin-60 (cpn60) sequences, with pairwise percent identities of 87.1-97.8% among the type strains. Pairwise co-occurrence patterns ofGardnerellaspp. in the vaginal microbiomes of 413 reproductive aged Canadian women were investigated, and several significant co-occurrences of species were identified. Abundance ofG. vaginalis, andswidsinskiiwas associated with vaginal symptoms of abnormal odour and discharge.Conclusionscpn60 barcode sequencing can provide a rapid assessment of the relative abundance ofGardnerellaspp. in microbiome samples, providing a powerful method of elucidating associations between these diverse organisms and clinical outcomes. Researchers should consider using cpn60 in place of 16S RNA for better resolution of these important organisms.