Background and Objective: Endoscopic third ventriculostomy is used to treat hydrocephalus. Different laser wavelengths have been proposed for laser-assisted endoscopic third ventriculostomies over the last decades. The aim of this study was to evaluate Thulium laser endoscopic third ventriculostomy heat penetration in the surrounding environment of the floor of the third ventricle in an in vitro setting with visualization of thermal distribution. Subsequently 106 Thulium laser endoscopic third ventriculostomy procedures were retrospectively analyzed to demonstrate safety. Methods: The in vitro visualization was based on the color Schlieren method. The heat penetration was measured beneath a tissue phantom of the floor of the third ventricle with a fiber of 365 μm in diameter at different energy settings; 1.0W (956 J/cm2), 2.0W (1,912 J/cm2), 4.0W (3,824 J/cm2), and 7.0W (6,692 J/cm2), with a pulse duration of 1.0 second. All experiments were repeated five times. In addition, 106 Thulium laser endoscopic third ventriculostomy procedures between 2005 and 2015 were retrospectively analysed for etiology, sex, complications, and laser parameters. Results: In the energy settings from 1.0 to 4.0 W, heat penetration depth beneath the phantom of the third ventricle did not exceed 1.5 mm. The heat penetration depth at 7 W, exceeded 6 mm. The clinical overall success rate was 80% at the 2-year follow-up study. Complications occurred in 5% of the procedures. In none of the 106 investigated clinical patients bleeding or damage to the basilar artery was encountered due to Thulium laser ablation. Conclusions: The in vitro experiments show that under 4.0W the situation is considered safe, due to low penetration of heat, thus the chance of accidentally damaging critical structures like the basilar artery is very small. The clinical results show that the Thulium laser did not cause any bleeding of the basilar artery, and is a safe technique for laser endoscopic third ventriculostomy. Lasers Surg. Med. 50:629–635, 2018. © 2017 Wiley Periodicals, Inc.