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In the UK, guidewires have traditionally been used for localization of non-palpable breast lesions in patients undergoing breast conservation surgery (BCS). Radioactive seed localization (RSL) using Iodine-125 seeds is an alternative localization method and involves inserting a titanium capsule, containing radioactive Iodine-125, into the breast lesion. We aim to demonstrate feasibility of RSL compared with guidewire-localization (GWL) for BCS in the UK. Methods: Data were collected on 100 patients with non-palpable unifocal invasive carcinoma of the breast undergoing GWL WLE prior to the introduction of RSL and the first 100 patients treated with RSL WLE. Statistical comparisons were made using Χ 2-squared analysis or unpaired two-sample t-test. Significance was determined to be at p ≤ 0.05. Results: Mean total tumour size was 19.44 mm (range: 5-55) in the GWL group and 18.61 mm (range: 3.8-59) in the RSL group (p = 0.548), while mean total specimen excision weight was significantly lower in the RSL group; 31.55 g (range: 4.5-112) vs 37.42 g (range: 7.8-157.1) (p = 0.018). Although 15 patients had inadequate surgical resection margins in the GWL group compared the 13 in the RSL group (15 vs 13%, respectively, p = 0.684), 10 of the patients in the GWL group had invasive carcinoma present resulting in at least one positive margin compared with only 3 patients in the RSL group (10 vs 3%, respectively, p = 0.045). Conclusion: In this study, RSL is shown to be noninferior to the use of GWL for non-palpable carcinoma in patients undergoing BCS and we suggest that it could be introduced successfully in other breast units. Advances in knowledge: Here we have demonstrated the use of RSL localization results in significant lower weight resection specimens of breast carcinoma when compared with a matched group using GWL, without any significant differences in oncological outcome between the groups.