Hot-iron branding is a common method of cattle identification. The branding procedure results in a burn scar that once healed, provides a permanent mark. Near infrared reflectance spectroscopy (NIRS) has been applied to monitor healing and determine age in cattle burn scars. The ability of NIRS to quantify histological characteristics of cattle burn scars has not been reported. The objective of this study was to evaluate the effectiveness of portable NIRS as a non-invasive chute-side diagnostic tool to detect histological changes in hot-iron brand burn scars during the healing process. Near infrared spectra (1100–1700nm) were obtained from burn scars on 15 Bos taurus cross steers (~ 270 d old; 238 ± 7 kg) at 21, 32, and 51 d post-branding. Spectra were obtained on branded (n = 3) and unbranded (n = 3) skin from each animal, each date. Skin punch biopsies (8 mm, n = 3 per animal) were obtained from a unique subset (n = 5) of animals at each date (n = 44 total). Spectra were analyzed as log 1/reflectance with 1st derivative and scatter correction applied. Skin tissue was preserved in ethanol and subjected to standard histological staining techniques at cutaneous, transitional, and sub-cutaneous layers. Relationships between spectra and histological values were determined by partial least squares regression. NIRS successfully predicted (P > 0.001) Gomori values for the total calibration (RSQ = 0.80; RMSE = 0.20). Success of Gomori calibrations varied by day of healing (d 21, NS; d 32, P > 0.001; d 51 NS). Other staining calibrations were also significant (P > 0.05) but not deemed useful for predicting quantitative histological values (RSQ > 0.5). Predictive ability generally declined with depth. Portable NIRS may prove useful as a chute-side histological diagnostic technique but requires further evaluation.