To evaluate the anatomy of the levator ani muscle in women with urogenital prolapse versus matched controls without prolapse using real-time two-dimensional (2-D) ultrasound. Prospective observational study. Tertiary referral urogynaecology unit. Forty-three women with pelvic organ prolapse (POP) and 24 women (controls) attending a gynaecology clinic without prolapse. All participants completed a standardised symptom questionnaire. The morphology of the vagina and paravaginal tissue was recorded at different levels. The thickness of the levator ani and the hiatal area were measured at rest. Reproducibility of the method was assessed by repeated measurements to assess intra-observer variability and inter-observer variability. This method showed good intra-observer and inter-observer reproducibility and reliability. In controls, the pubococcygeus muscle showed more regular echogenicity with no evidence of trauma, whereas in women with prolapse the muscle had mixed echogenicity. (P = 0.002). The mean thickness of the pubococcygeus did not differ between groups. The levator hiatal area was significantly larger in women with pelvic floor prolapse versus controls (17.8 cm2 versus 13.5 cm2, P < 0.001). This increase in hiatal area positively and significantly correlated with prolapse severity (P < 0.001). Morphology and hiatal area can be reliably imaged using 2-D ultrasound. Prolapse was related to changes in pelvic floor morphology and increased levator hiatal area. The use of 2-D ultrasound provides an important insight into the pathophysiology of prolapse.