Preoperative localization is important to optimize the surgical treatment of breast lesions, especially in nonpalpable lesions. Radioactive seed localization (RSL) using iodine-125 is a relatively new approach. To provide accurate guidance to surgery, it is important that the seeds do not migrate after placement. The aim of this study was to assess short-term and long-term seed migration after RSL of breast lesions. In 45 patients, 48 RSL procedures were performed under ultrasound or stereotactic guidance. In the first 12 patients, the lesion was localized with two markers: an iodine-125 seed and a reference marker. In 33 patients, 36 RSL procedures were performed using a single iodine-125 seed. All patients received control mammograms after seed placement and prior to surgery. In the patients with two markers, migration was defined as the difference in the largest distance between the markers observed in the mammograms. For single-marked lesions, migration was assessed by comparing distances between anatomical landmarks in the mammograms. RSL was successful in all patients. Seeds were in-situ for 59.5 days on average (3-136 days). The detection rate during surgery was 100%. Overall, an average seed migration of 0.9 mm (standard deviation 1.0 mm) was observed. Neither differences in lesion type, nor days in situ, type of surgery or radiologic localization method were found to have impact on seed migration. RSL is an accurate preoperative localization method for breast lesions with negligible seed migration, independent of time in-situ.