AIMS: Inadvertent puncture of the aortic root (AR) is a well-known complication of transseptal puncture (TSP). Strategies for handling of this potentially lethal complication have not been identified yet. In this study, we present typical anatomical locations and clinical management of aortic root puncture (ARP) due to TSP. METHODS AND RESULTS: All patients with ARP were retrospectively collected from seven hospitals. Aortic root puncture was identified and classified regarding angiographical and intraoperative findings in cardiac surgery: (i) TSP from the right atrium (RA) to the non-coronary sinus (NCS), (ii) TSP from RA to the non-coronary sinutubular junction (STJ), and (iii) TSP from RA to the ascending aorta (AA). A total of 24 patients with inadvertent ARP were identified. In 19 patients, penetration of the aorta was accomplished by the inner dilator, in 5 patients by the complete sheath. Previous cardiac surgery had been performed in six patients. There were 13 RA-to-NCS punctures, 2 RA-to-STJ punctures, and 9 RA-to-AA punctures. No cardiac tamponade (CT) occurred in patients with RA-to-NCS and RA-to-STJ punctures. In 8 of 9 patients with RA-to-AA puncture, CT occurred immediately requiring urgent pericardiocentesis and surgical repair. Two patients died after surgical repair. In the 16 patients without surgical therapy, no shunt from the AR to the RA was observed 3 months after the procedure. CONCLUSION: Aortic root puncture due to mislead TSP via NCS or STJ is usually not associated with a severe clinical course while ARP into the AA via the epicardial space generally leads to CT requiring surgical repair. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]