Significance: Burn injuries represent a global public health problem that kills an estimated 180,000 people annually. Non-fatal burns result in prolonged hospitalization, disfigurement, and disability. The most common, convenient, and widely used method for assessing burn depth is physical or visual examination, but the accuracy of this method is reportedly poor (60% to 75%). Rapid, correct assessment of burn depth is very important for the optimal management and treatment of burn patients. New methods of burn depth assessment that are inexpensive, simple, rapid, non-contact, and non-invasive are therefore needed. Aim: The aim of this study was to propose an approach to visualize the spatial distribution of burn depth using hemoglobin parameters estimated from spectral diffuse reflectance imaging and to demonstrate the feasibility of the proposed approach for differentiating burn depth in a rat model of scald burn injury. Approach: The new approach to creating a spatial map of burn depth was based on canonical discriminant analysis (CDA) of total hemoglobin concentration, tissue oxygen saturation, and methemoglobin saturation as estimated from spectral diffuse reflectance images. Burns of three different degrees of severity were created in rat dorsal skin by 10-s exposure to water maintained at 70°C, 78°C, and 98°C, respectively. Spectral images for dorsal regions were acquired under anesthesia immediately after burn injury and at 24 h, 48 h, and 72 h after injury. Results: Most areas of images in the group with skin exposed to 70°C water and 98°C water were classified as 70°C burn and 98°C burn, respectively. In contrast, no significant difference between areas classified as 78°C burn and 98°C burn from 24 h to 72 h was evident in the group with skin exposed to 78°C water, suggesting that burn depth was heterogeneous. Conclusions: The proposed approach combining diffuse reflectance spectral imaging and CDA appears promising for differentiating 70°C burns from 78°C burns and 98°C burns, and 98°C burns from 70°C burns and 78°C burns at 24 to 72 h after burn injury in a rat model of scald burn injury.