ABSTRACT To our knowledge, no data are available on whether the microbial species composition and abundance sampled with self-collected vaginal swabs are comparable to those of swabs collected by clinicians. Twenty healthy women were recruited to the study during a routine gynecological visit. Eligible women were between 18 and 40 years old with regular menstrual cycles. Participants self-collected a vaginal swab using a standardized protocol and then were examined by a physician, who collected an additional five swabs from the lateral wall of the mid-vagina. In this study, the self-collected and three physician-obtained swabs were analyzed and compared using terminal restriction fragment length polymorphism and sequence analyses of the 16S rRNA genes. Vaginal microbial community comparative statistical analyses of both T-RFLP and 16S rRNA gene sequence datasets revealed that self-collected vaginal swabs sampled the same microbial diversity as physician collected swabs of the mid-vagina. These findings enable large-scale, field-based studies of the vaginal microbiome.