Gardnerella vaginalis is an important component of the human vaginal microflora. It is proposed to play a key role in the pathogenesis of bacterial vaginosis (BV), the most common vaginal condition. Here we describe the development, validation and comparative analysis of a novel molecular approach capable of G. vaginalis identification, quantification and subtyping in noncultured vaginal specimens. Using two quantitative PCR (qPCR) assays, we analysed G. vaginalis bacterial loads and clade distribution in 60 clinical vaginal-swab samples. A very high pathogen prevalence was revealed by species-specific qPCR not only among BV patients (100 %), but also in healthy women (97 %), although the G. vaginalis concentration was significantly lower in non-BV samples. G. vaginalis clades identified in vaginal specimens by subtyping multiplex qPCR, which targets four clade-specific genetic markers, had frequencies of 53 % for clade 1, 25 % for clade 2, 32 % for clade 3 and 83 % for clade 4. Multiple clades were found in 70 % of samples. Single G. vaginalis clades were represented by clade 1 and clade 4 in 28 % of specimens. A positive association with BV was shown for clade 1 and clade 3, while clade 2 was positively associated with intermediate vaginal microflora, but not with BV. Clade 4 demonstrated no correlation with the disorder. The presence of multiple clades had a high positive association with BV, whereas G. vaginalis identified as a single clade was negatively linked with the condition. Polyclonal G. vaginalis infection may be a risk factor for BV.