Introduction: Impalpable breast cancers require precise pre-operative lesion localisation to minimise re-excision rates. Conventional techniques include hookwire insertion using stereotactic guidance. Newer techniques include the use of tomosynthesis guidance and the use of iodine-125 seeds. This study compares the accuracy of lesion localisation with hookwire or seed insertion using prone stereotactic or upright tomosynthesis guidance. Methods: This registered quality improvement activity did not require formal ethics approval. The post-localisation images for 116 lesions were reviewed. The distance from the lesion or breast biopsy marker to the hookwire or seed was measured on post-insertion mammograms. The relative placement accuracy of hookwire or seed using prone stereotactic or upright tomosynthesis guidance was compared. A lesion to seed or wire distance > 10 mm was considered technically unsatisfactory. Results: 94.8% of the seeds and wires inserted via prone stereotactic guidance were accurately placed, compared with 89.6% of those inserted via upright tomosynthesis. There were twice as many technically unsatisfactory insertions under upright tomosynthesis guidance. The majority of the unsatisfactory insertions using upright tomosynthesis occurred when the lesion was at or below the level of the nipple and the insertion was performed craniocaudally. Conclusion: The degree of accuracy of pre-operative localisation of impalpable breast lesions is significantly higher with the use of prone stereotactic rather than upright tomosynthesis guidance. This was most evident with the placement of I-125 seeds, and in cases where the target lesion was located below the level of the nipple.