The masticatory muscles of the horse are arranged in an asymmetrical pattern. Four individual muscles on the medial side of the mandible are opposed by one muscle (M. masseter) on the lateral side. However, recent studies on various herbivorous mammals indicate that the masseter muscle features a complex stratigraphic structure that might account for a functional diversity resembling an arrangement of several individual muscles. The functional consideration of the multidirectional equine masticatory movements leads to a similar hypothesis. In order to elucidate the detailed anatomy of the equine masseter muscle, eight cadaveric equine heads were dissected. Additional 29 skull specimens were assessed with regard to the masseteric attachment within the Fossa masseterica. A constant arrangement of nine individual muscular layers within the masseteric muscle was determined. The individual layers were clearly separated by tendon plates and their attachment areas at the masseteric fossa of the mandible were arranged in a constant pattern of bony ridges. With similar consistency, the main trunk of the masseteric nerve was found to run from dorsocaudal to ventrorostral: On that course, the nerve penetrated between the muscular layers from medial to lateral at constant positions. The findings of this study serve as a basis for further studies with the aim of developing biomechanical concepts of equine masticatory movement.