Objective To identify Mobiluncus species with matrix-assisted laser desorption/ioniza-tion time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing and to analyze anti-biotic resistance genes and phylogenetic relationships among Mobiluncus species with whole genome sequen-cing. Methods Twenty-five Mobiluncus strains were isolated after anaerobic culture of 65 vaginal secretion samples of patients with bacterial vaginosis (BV) and identified to species level by MALDI-TOF MS and 16S rRNA gene sequencing. The whole genome DNA of each strain was extracted for next-generation sequencing. SPAdes was used to assembly genomes. Resistance genes were searched in ARGD database. The core ge-nome of all isolates was analyzed by Harvest to construct phylogenetic tree. Results MALDI-TOF MS could only identify Mobiluncus curtisii, while 16S rRNA gene sequencing could identify both Mobiluncus mulieris and Mobiluncus curtisii. Results of the whole genome sequencing showed that tetracycline resistance gene tet ( o) and macrolides resistance gene erm(x) were the two predominant acquired resistance genes of Mobilun-cus with a positive rate of 84. 7% and 61. 5% respectively. Intra-species relationships of the two Mobiluncus species were close, but a distant phylogenetic relationship was found between the two species. Conclusion This study shows that MALDI-TOF MS can't be used to identify Mobiluncus mulieris at present. Mobiluncus strains have potential resistance to tetracycline and macrolides. Intra-species evolution of Mobiluncus is slow, which indicates that there is no growing trend towards new species for the time being.