A newly developed Gamma Knife relocatable eXtend frame system has enabled the delivery of multi-session Gamma Knife radiosurgery without the use of skull pin fixation frame system. In this study, we investigate and report for the first time the whole procedural radiological accuracy for administering such treatments. To quantify the radiological alignment, the commonly used Winston-Lutz test was modified and used to determine the device accuracy of the eXtend frame system. Patient setup uncertainties relative to the device were further measured for a series of treatment sessions (n = 58), and then incorporated with the Winston-Lutz test results from individual patient-specific eXtend frame systems. The whole-procedure mean 3D radiological setup uncertainty was determined to be 0.69 ± 0.73 mm (1δ) from all the cases analyzed, and the mean 90% confidence level margins were found to be 0.55, 0.78 and 0.72 mm along the x-, y-, and z-axis, respectively. Our results therefore demonstrated that sub-millimetric radiological accuracy is clinically achievable for multi-session Gamma Knife radiosurgery treatments and a 1 mm margin along the major axes is sufficient for planning multi-session Gamma Knife radiosurgery treatments.