Fresh turkey meat color is determined by many factors that include muscle fiber type composition and heme protein concentrations. These factors either are affected by or influence biochemical events occurring postmortem. Deviations in the processing environment also can result in aberrant fresh meat quality and may ultimately change the quality characteristics of further processed products. Our objective was to describe the underlying cause and significance of the two-toning color defect in fresh turkey breast. In the first experiment, pectoralis major muscles were collected, classified as single- or two-toned, and analyzed using image processing to characterize fresh turkey color. Samples from the large and small lobes of the pectoralis major muscle were collected for pH, glycolytic intermediates, protein abundance, mRNA expression, and quality characteristics. In the second experiment, time from stun to exsanguination was tested as a promoter of fresh turkey color. Results from the first experiment showed that the turkey breast possesses two distinct lobes. The large lobe had greater (P < 0.05) glycolytic potential, lactate content, lactate dehydrogenase (LDH) abundance, and centrifugal drip loss, while pH, myoglobin mRNA expression, and soluble protein levels were lower (P < 0.05) compared to the small lobe. Results from the second experiment showed that reducing time from stun to exsanguination enhanced (P < 0.05) fresh turkey color by mitigating the differences between the two lobes. Our results also showed that birds exsanguinated first had greater (P < 0.05) muscle pH values and body temperatures. These results show inherent differences in breast muscle and processing conditions interact to establish variations in fresh turkey color.