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BACKGROUND: No objective technique exists to distinguish necrotic from viable tissue, risking over-excision in burns and loss of wound healing potential. Second Window Indocyanine Green (SWIG) is a novel fluorescence-imaging modality being studied to identify residual solid tumors during oncological surgery. SWIG has also been shown to have avidity for necrosis in animal models but translation of these findings to humans is lacking. The objective of this study was to evaluate SWIG in the identification of burn wound necrosis, and compare it to previously published indocyanine green angiography (ICGA) techniques. STUDY DESIGN: This study utilized mouse, human skin xenograft and human patient burn models. Brightfield and SWIG near-infrared (NIR) imaging were performed on macroscopic tissue samples, which were then cryopreserved, sectioned and analyzed for microscopic fluorescence. SWIG fluorescence findings were correlated to visual assessment of the burn wound, as well as histological markers of necrosis using hematoxylin and eosin (H&E) and lactate dehydrogenase stains (LDH). RESULTS: We found that SWIG identified burn necrosis in a manner dependent on the dose and timing of indocyanine green (ICG) administration and had an inverse fluorescence signal compared to ICGA. Furthermore, SWIG fluorescence identified the interface of viable and non-viable tissue. CONCLUSION: Our study confirmed that ICGA is an inconsistent and non-standardized modality to evaluate burn injuries. In contrast, SWIG imaging is a potential imaging modality to objectively prognosticate burn wound healing potential and guide intraoperative burn excision. Further studies are needed to define ratios of fluorescence intensity values to guide surgical decision-making in burn excision, and to better define how ICG is retained in necrotic tissue to enhance utility of SWIG in other disease processes.