Introduction: Sentinel lymph node biopsy (SLNB) in cutaneous melanoma (CM) is performed to identify patient at risk of regional and distant relapse. We hypothesized that timing of lymphoscintigraphy may influence the accuracy of SLNB and patient outcomes. Methods: We reviewed prospective data on patients undergoing SLNB for CM at a large university cancer-center between 2008-2015, examining patient and tumor demographics and time between lymphoscintigraphy (LS) and SLNB. Kaplan-Meier survival analysis assessed disease-specific (DSS) and overall-survival (OS), stratified by timing of LS. Cox multivariate regression analysis assessed independent risk factors for survival. Results: We identified 1015 patients. Median follow-up was 45 months (IQR 26-68 months). Univariate analysis showed a 6.8% absolute DSS (HR 1.6 [1.03-2.48], p= 0.04) benefit and a 10.7% absolute OS (HR 1.64 [1.13-2.38], p=0.01) benefit for patients whose SLNB was performed < 12 hrs of LS (n= 363) compared to those performed >12 hours (n=652). Multivariate analysis identified timing of LS as an independent predictor of OS (p=0.007) and DSS (p=0.016) when competing with age, sex, Breslow thickness (BT) and SLN status. No difference in nodal relapse rates (5.2% v 4.6%; p=0.67) was seen. Both groups were matched for age, sex, BT and SLN status. Conclusion: These data have significant implications for SLNB services, suggesting delaying SLNB >12 hours after LS using a Tc99-labelled nanocolloid has a significant negative survival impact for patients and should be avoided. We hypothesise that temporal tracer migration is the underlying cause and advocate further trials investigating alternative, 'stable' tracer-agents.