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Abstract Chlamydia trachomatis, Mycoplasma spp., Neisseria gonorrhoeae and Treponema pallidum are sexually transmitted pathogens that threaten reproductive health worldwide. In this study, vaginal swabs obtained from women (n = 133) that attended an infertility clinic in China were tested with qPCRs for C. trachomatis, Mycoplasma spp., N. gonorrhoeae, T. pallidum and tetracycline resistance genes. While none of vaginal swabs were positive for N. gonorrhoeae and T. pallidum, 18.8% (25/133) of the swabs were positive for Chlamydia spp. and 17.3% of the swabs (23/133) were positive for Mycoplasma species. All swabs tested were positive for tetracycline resistance gene tet(M) which is the most effective antibiotic for bacterial sexually transmitted infections. The qPCRs determined that the gene copy number per swab for tet(M) was 7.6 times as high as that of C. trachomatis 23S rRNA, and 14.7 times of Mycoplasma spp. 16S rRNA. In China, most hospitals do not detect C. trachomatis and Mycoplasma spp. in women with sexually transmitted infections and fertility problems. This study strongly suggests that C. trachomatis and Mycoplasma spp. should be routinely tested in women with sexually transmitted infections and infertility in China, and that antimicrobial resistance of these organisms should be monitored. Further studies are warranted to determine the prevalences in different regions and associated risk factors.