Beef colour is essential to consumer acceptability with dark muscle colours being problematic. Dark meat has less light scattering but the mechanisms are unknown. We hypothesise that three mechanisms are responsible for decreased light scattering in dark meat, namely (i) larger lateral separation of myofilaments, (ii) decreased optical protein density in the I-band and (iii) decreased denaturation of sarcoplasmic proteins. Nineteen beef longissimus thoracis muscles, divided into ‘light’, ‘medium’ and ‘dark’ colour groups, were assessed for light scattering by reflectance confocal microscopy, sarcomere length, and myofilament lattice spacing by small-angle X-ray diffraction. Dark muscles had a longer lattice spacing, shorter sarcomeres and wider muscle fibre diameters compared to lighter colour groups, indicating that the transverse spacing of muscle fibres that occurs post-mortem, with pH decline, is central to light scattering development. Dark muscles also had more degradation of the Z-disc and higher sarcoplasmic protein activities, which could impact on the optical density and contribute to lower light scattering.