Objectives: Pouch of Douglas (POD) obliteration can be predicted with a high degree of certainty and reproducibility using the dynamic transvaginal ultrasound (TVS) sliding-sign technique. So far, studies on POD obliteration prediction have focused on tertiary-care populations with high prevalence of endometriosis; however, POD obliteration may exist in individuals with asymptomatic endometriosis or other conditions. Our primary aim was to determine the prevalence of a negative sliding sign, representing POD obliteration, in a cohort of patients undergoing TVS for any gynecological indication. Methods: This was a prospective observational study of consecutive women with an indication for gynecological TVS, conducted at a high-volume ultrasound practice between July and August 2018. Clinical and surgical history, indication for TVS and TVS findings were documented. The prevalence of TVS-confirmed POD obliteration, determined by interpretation of the sliding sign, was calculated for the entire cohort and for the subgroups of women with and without risk factors for endometriosis. High risk for endometriosis was defined as having (1) a TVS referral for endometriosis-like pelvic pain or endometriosis specifically and/or (2) clinical symptoms or signs suggestive of endometriosis. Low risk was defined as the absence of these characteristics. Results: During the study period, 1043 consecutive women underwent TVS. After excluding those who underwent transabdominal ultrasound, had a history of hysterectomy or with missing data, 909 women were analyzed. The prevalence of a negative sliding sign in the entire cohort was 47/909 (5.2%). A negative sliding sign was observed in 22/639 (3.4%) women with a low risk for endometriosis and 25/243 (10.3%) of those with a high risk for endometriosis (difference in proportions, 6.9% (95% CI 2.8–10.9%); P < 0.001). Conclusions: We have demonstrated an overall prevalence of a negative sliding sign, suggesting POD obliteration, of 5.2% (or 1/20) in women seeking TVS for a gynecological indication. The prevalence of negative sliding sign in low-risk women is not negligible (3.4% or 1/29 women). These women are most likely to have asymptomatic endometriosis or another important etiology of POD obliteration. The prevalence of a negative sliding sign is approximately three-times higher in women with signs and/or symptoms of endometriosis (10.3% vs 3.4%).