Considering the variability of bay coat colour, we aimed to investigate the association of different shades of bay with ASIP and MC1R genotype combinations and we studied the discrimination between the bay and black coat colour. We phenotypically characterized coat colour using a spectrophotometer. The measurements were based upon international standards as defined by the CIE L*a*b* colour system and we phenotyped five different body parts (neck, armpit area, shoulder, belly, croup) of 43 bay and 14 black horses kept under standardized conditions. From the five measuring points a stepwise discriminant analysis revealed that chromacity and luminescence of armpit area and luminescence of the neck were the most important traits to differentiate between black and bay horses, whereas it was shown that the red colour spectrum of neck, luminescence of the neck, and luminescence of the armpit area grouped bay horses according to their ASIP and MC1R genotype combinations. Within the group of bay horses the analyses identified a single ASIP/MC1R genotype combination (A/a E/E) where colour variables differed significantly from the three remaining genotype groups. A/a E/E horses were characterized in all body parts except in the armpit region by significant darker shades (lower luminescence, less chromacity). Regarding classifications of coat colour, we found in the significant duster of A/a E/E horses the coat colour categories seal brown and dark (mahogany) bay. Overall, we were able to show that the characterization of equine coat colour based upon international standards as defined by the CIE L*a*b* colour system represents a valuable tool for a precise description of colour variation and association analyses.