Objective <p>To evaluate the diagnostic performance of combined transscrotal–transrectal ultrasonography in predicting sites of obstructive azoospermia.</p> Materials and methods <p>From June 2019 to March 2023, 166 obstructive azoospermia patients who underwent surgical exploration were enrolled in the retrospective study. The data of combined transscrotal–transrectal ultrasonography in 166 patients were collected and analyzed. The receiver operating characteristic (ROC) curve analysis was employed to evaluate the diagnostic performance of these ultrasonographic measurements for localizing different obstructive sites.</p> Results <p>There were 9 sides of intratesticular obstruction, 239 sides of epididymal obstruction, 68 sides of vas deferens obstruction, and 16 sides of ejaculatory duct obstruction. The sensitivity, specificity, and the area under the curve (AUC) for combined transscrotal–transrectal ultrasonography were 44.4%, 98.5% and 0.714 for diagnosing intratesticular obstruction; 97.9%, 84.9% and 0.919 for diagnosing epididymal obstruction; 82.4%, 99.2% and 0.913 for diagnosing vas deferens obstruction; and 87.5%, 99.1% and 0.93 for diagnosing ejaculatory duct obstruction. The sensitivity, specificity, and AUC were 88.9%, 83.9% and 0.842 in diagnosing intratesticular obstruction for a rete testis thickness cut-off of 3.0 mm; 81.0%, 100% and 0.949 in diagnosing vas deferens obstruction for a 0.8 mm cutoff for the internal diameter of the scrotal section of the vas deferens; and 62.5%, 92.6% and 0.769 in diagnosing ejaculatory duct obstruction for a seminal vesicle diameter cut-off of 12.5 mm.</p> Conclusion <p>Combined transscrotal–transrectal ultrasonography, evaluating specific structures of rete testis thickness, seminal vesicle diameter, and the internal diameter of the scrotal vas deferens, could accurately localize obstruction sites in obstructive azoospermia patients.</p> Critical relevance statement <p>Combined transscrotal–transrectal ultrasonography demonstrated high diagnostic performance in predicting the sites of epididymal, vas deferens, and ejaculatory duct obstruction in patients with obstructive azoospermia.</p> Key Points <p><UnorderedList Mark="Bullet"> <ItemContent> <p>The diagnostic performance of combined transscrotal–transrectal ultrasonography in obstructive azoospermia was evaluated.</p> </ItemContent> <ItemContent> <p>Ultrasound measurements of specific structures significantly improve the prediction of obstruction sites.</p> </ItemContent> <ItemContent> <p>Combined transscrotal–transrectal ultrasonography accurately localizes obstruction sites in obstructive azoospermia patients.</p> </ItemContent> </UnorderedList></p> Graphical Abstract <p></p>