<b><i>Introduction:</i></b> The intersection of Bejjani’s line with the well-delineated medial subthalamic nucleus (STN) border on MRI has recently been proposed as an individualized reference in subthalamic deep brain stimulation (DBS) surgery for Parkinson’s disease (PD). We, therefore, aimed to investigate the applicability across centers of the medial STN border as a patient-specific reference point in STN DBS for PD and explore anatomical variability between left and right mesencephalic area within patients. Furthermore, we aim to evaluate a recently defined theoretic stimulation “hotspot” in a different center. <b><i>Methods:</i></b> Preoperative 3-Tesla T2 and susceptibility-weighted images (SWI) were used to identify the intersection of Bejjani’s line with the medial STN border in left and right mesencephalic area. The average stereotactic coordinates of the center of stimulation relative to the medial STN border were compared with the predefined theoretic stimulation “hotspot.” <b><i>Results:</i></b> Fifty-four patients provided 108 stereotactic coordinates of medial STN borders on both sequences. Significant difference in means was found in the Y-(anteroposterior) and Z-(dorsoventral) directions (T2 vs. SWI; <i>p</i> &#x3c; 0.001). Mean coordinates in the Y-(anteroposterior) direction differed significantly between left and right mesencephalic area (T2: <i>p</i> &#x3c; 0.001; SWI: <i>p</i> = 0.021). Sixty-six DBS leads were placed in 36 patients that had finished stimulation programming, and the average stereotactic coordinates of the center of stimulation relative to the medial STN border on T2 sequences were 3.1 mm lateral, 0.7 mm anterior, and 1.8 mm superior, in proximity of the predefined theoretic stimulation “hotspot.” <b><i>Conclusion:</i></b> The medial STN border is applicable across centers as a reference point for STN DBS surgery for PD and seems suitable in order to account for interindividual and intraindividual anatomical variability if one is aware of the discrepancies between T2-weighted imaging and SWI.