Objective: To correlate with histopathological features the adenomyosis‐induced morphological alterations of the outer myometrium and the inner myometrium (‘junctional zone’, JZ) detectable on two‐ (2D) and three‐dimensional (3D) transvaginal ultrasound imaging (TVS), and to evaluate their diagnostic accuracy for adenomyosis.Methods: Premenopausal patients scheduled for hysterectomy for benign pathology were enrolled in this prospective study. Before hysterectomy all patients underwent detailed 2D‐TVS and 3D volume acquisition of the entire uterus. The major sonographic signs of adenomyosis were noted. On the multiplanar coronal and longitudinal views obtained by 3D‐TVS we measured the maximum and minimum JZ thickness from the basal endometrium to the internal layer of the outer myometrium (JZmax, JZmin), the difference between them (JZdif = JZmax − JZmin) and the ratio JZmax/total maximum myometrial thickness. Results of these examinations were correlated blindly to the presence of adenomyosis on histological specimens.Results: A total of 72 premenopausal patients underwent 2D‐ and 3D‐TVS before hysterectomy. The histological prevalence of adenomyosis was 44.4% (32/72 patients). In diagnosing adenomyosis, the presence of myometrial cysts was the most specific 2D‐TVS feature (specificity, 98%; accuracy, 78%) and heterogeneous myometrium was the most sensitive (sensitivity, 88%; accuracy, 75%). The 3D‐TVS markers JZdif ≥ 4 mm and JZ infiltration and distortion had high sensitivity (88%) and the best accuracy (85% and 82%, respectively). For 2D‐TVS and 3D‐TVS, respectively, the overall accuracy for diagnosis of adenomyosis was 83% and 89%, the sensitivity was 75% and 91%, the specificity was 90% and 88%, the positive predictive value was 86% and 85% and the negative predictive value was 82% and 92%.Conclusions: The coronal section of the uterus obtained by 3D‐TVS permits accurate evaluation and measurement of the JZ, and its alteration has good diagnostic accuracy for adenomyosis. Copyright © 2011 ISUOG. Published by John Wiley & Sons, Ltd.