© 2021, The Author(s), under exclusive licence to Springer-Verlag France SAS, part of Springer Nature.Purpose: Cervical dystonia is a common movement disorder for which botulinum toxin (BoNT) is the first choice treatment. Injecting the specific neck muscles can be challenging because of their thin morphology and deep locations. We, therefore, designed a study to investigate the locations of the posterior neck muscles to help the physician predict the locations of the targeted neck muscles and to protect the vertebral vessels from injury during deep injections. Methods: The posterior neck region was divided into four quadrants by imaginary lines passing vertically and transversely through the spinous process of C2 vertebra (C2sp). The thicknesses and depth of the posterior neck muscles were measured in ten formaldehyde-fixed adult male cadavers. These muscles were located and a projection of them was drawn on the neck. Using the measurements, colored latex in place of BoNT was injected into them in one cadaver. The cadaver was dissected to investigate whether the muscles were colored. Results: 2 cm above the C2sp, trapezius, splenius capitis (SPC) and semispinalis capitis (SSC) were colored at depths of 10.70 mm, 11.88 mm and 15.91 mm, respectively. 2 cm below the C2sp, the trapezius, SPC and SSC were colored at depths of 20.89 mm, 23.25 mm and 27.63 mm, respectively. The posterior neck muscles were had taken up their assigned colors when they were injected according to the results obtained in this study. The vertebral vessels were not colored. Conclusions: Although BoNT injection into the posterior neck muscles is challenging, we think that it can be practically and safely applied using the measurements obtained in this study.