The aim of this study was to determine intramuscular distribution pattern of the masseteric nerve, thus providing information regarding the most efficient and safe site for botulinum toxin (BTX) injection for conventional BTX intervention therapy in the treatment of masseteric hypertrophy.Twelve masseter muscles were dissected, and the pattern of innervation of the masseteric nerve was observed in the superficial, middle, and deep layers. We also analyzed 10 muscles that had been stained using Sihler's staining technique.The nerve branches from the posterosuperior and posteroinferior groups innervating the deep and middle layers of the masseter muscle, respectively. Among the nerve twigs originating from the anteroinferior nerve group, 2 or 3 perforated the superficial layer of the muscle. Observation of stained specimens revealed that all perforating branches innervating the superficial layer were mainly confined to and distributed within areas V or VI.Between 2 and 4 perforating branches supply the superficial layer of the masseter muscle. In addition, the richest arborization of the perforating masseteric nerve branches is confined mostly to area V, approximately in accordance with the BTX injection point that is currently used clinically. Area V is thus strongly recommended as the most efficient and safe BTX injection area for the treatment of masseteric hypertrophy