BACKGROUND: Despite the positive effect of botulinum neurotoxin (BoNT) injections in thoracic outlet syndrome (TOS) treatment, there is insufficient anatomical evidence of its use in the anterior scalene (AS) and middle scalene (MS) muscles. OBJECTIVES: This study aimed to provide safer and more effective guidelines for the injection of botulinum neurotoxin into scalene muscles for the treatment of thoracic outlet syndrome. STUDY DESIGN: The study was based on an anatomical study and ultrasound studies. SETTING: This study was conducted at the Division in Anatomy and Developmental Biology, Department of Oral Biology, Human Identification Research Institute, BK21 FOUR Project, Yonsei University College of Dentistry (Seoul, Republic of Korea) METHODS: Ten living volunteers underwent ultrasonography, and the depths of the anterior scalene (AS) and middle scalene (MS) muscles were calculated from the skin surface. In cadaveric specimens, fifteen AS and 13 MS muscles were stained using the Sihler staining procedure; the neural arborization pattern was identified, and localized dense portions were investigated. RESULTS: The mean depth of the AS was 9.19 ± 1.56 mm, and that of the MS was 11.64 ± 2.73 mm at 1.5 cm above the clavicle. At 3 cm above the clavicle, the AS and MS were clearly located 8.12 ± 1.90 mm and 10.99 ± 2.52 mm deep, respectively. The nerve ending points were highest in the lower three-quarters of the AS (11/15 cases) and MS muscles (8/13 cases), followed by the lower quarter (AS muscle, 4/15 cases; MS muscle, 3/13 cases). LIMITATIONS: There are many difficulties for clinics to directly perform ultrasound-guided injections in clinical practice. However, results of this study can be used as basic data. CONCLUSION: According to anatomical features, the appropriate location for botulinum neurotoxin injection in the AS and MS muscles for the treatment of TOS is the lower portion of the scalene muscles. Therefore, it is recommended to inject at a depth of approximately 8 mm for AS and 11 mm for MS at a point 3 cm above the clavicle. KEY WORDS: Thoracic outlet syndrome, ultrasound-guided injection, nerve arborization pattern, botulinum neurotoxin, noninvasive treatment, scalene muscles, anterior scalene, middle scalene