AbstractTrepanation skulls have been found clustered in the Gan‐Qing region of northwestern China, where the trepanation also demonstrated more signs of healing than those from other areas. In this study, two skulls belonging to the pre‐Qiang people from the Mapai cemetery in the late Neolithic period in the Gan‐Qing region with trepanation were observed using ultra‐depth electron microscopy and CT imaging. Moreover, trepanation experiments were conducted to test different tools and tool combinations to investigate the trepanation technique used by the pre‐Qiang people. Results demonstrated that the trepanation openings with signs of healing were the work of experienced trepanation practitioners, most likely for therapeutic purposes. Our experiments also demonstrated that different tools need to be used to generate similar perforations, suggesting a mature trepanation procedure by the pre‐Qiang people. Although it seems clear that the two individuals in this study underwent neurosurgical operations, it is impossible to determine why that surgery was performed. The trepanation phenomenon of the Gan‐Qing region enriches our knowledge of lifestyles, health status, and primitive yet then advanced medical techniques and healthcare.