Background Preoperative localization of the parathyroid gland prior to a minimally invasive parathyroidectomy (MIP) is important because of varying locations of the parathyroid gland. Several methods have been described to localize the affected gland. One novel technique is the use of an iodine 125 (I-125) seed as a marker. The aim of this study is to evaluate the feasibility of using an I-125 seed in localizing the diseased parathyroid gland prior to MIP. Materials and methods This is a pilot study of 10 patients performed in the Amphia Hospital, the Netherlands. Patients in whom primary hyperparathyroidism (PHPT) was diagnosed in combination with 1 enlarged parathyroid gland on ultrasound (US) and scintigraphy and who were eligible for MIP were included in this study. These patients underwent a preoperative US-guided I-125 seed placement in the affected parathyroid gland. The main study parameters were the feasibility of the placement, intraoperative localization of the diseased gland and complications. Results A total of 10 patients were included. The US-guided I-125 placement in the affected parathyroid gland was technically feasible in the majority of cases. Because of the anatomical location of the gland, the placement was difficult in 2 patients, resulting in suboptimal position and possible misplacement of the marker. MIP was uncomplicated in most cases. Complications during surgery were mainly intraoperative bleeding. Conclusions The use of an I-125 seed for preoperative localization in PHPT is a relatively safe technique in parathyroid surgery. More research is needed to compare this technique with other preoperative localization techniques.