To test the hypothesis that sub-areolar (SA) lymphoscintigraphy (LSG) identifies the same sentinel node as peri-tumoural (PT) injections. It is commonly believed that all LSG techniques will identify the same sentinel lymph nodes (SLN) draining the breast. Hybrid imaging technology (SPECT/CT) allows accurate identification of the exact location of SLNs. Using SPECT/CT SA and PT LSG techniques were compared. In a multi-centre trial 39 patients sequentially underwent LSG (SA followed by PT) separated by 2-7 days. Patients were referred by 4 surgeons to 3 LSG centres, with standardization of isotope (99mTc-antimony sulfide colloid), LSG and SPECT/CT evaluation techniques. LSG were evaluated for SLN concordance and degree of discordance in the axilla and internal mammary nodes (IMN). 39 eligible patients, median age 62 years, were recruited. Successful axillary SLN mapping for SA and PT injection techniques was 87% and 95% respectively. Successful internal mammary SLN mapping occurred with SA and PT LSG in 5% and 36% respectively. Discordance was identified in the IMN (39%) and axilla (21%), with an overall rate of discordance between SA and PT LSG of 56%. There is a high level of discordance in the localization of SLN by these commonly used LSG injection techniques. This discordance has implications for accuracy of axillary and extra-axillary staging and could impact on patient outcome.