Most studies have focused on myoglobin regarding meat colour development, with little focus on the contribution of muscle structure and light scattering. Our aim was to investigate the pH-dependent changes in muscle structure, on the light scattering properties of the meat. Beef longissimus thoracis muscles were segregated into light, medium or dark colour groups (n = 18). ‘Dark’ muscles had a high ultimate pH (pHu), lower lightness (L*), redness (a*), higher myoglobin concentration and deoxymyoglobin content compared to other muscles. Reflectance confocal laser scanning microscopy (rCLSM) revealed ‘dark’ muscles had decreased global brightness (indicator of light scattering) and increased fibre width. In a secondary experiment, decreasing the homogenisation buffer pH from 6.10 to 5.40 induced a 17% shrinkage in high pHu muscle fibres, which increased light scattering by ~ 25%. In conclusion, rCLSM demonstrated that muscle structure contributes to the magnitude of light scattering by a pH dependent mechanism.