The purpose of the present study is to provide useful data for anatomists and clinicians by describing the topography of the palmaris brevis muscle, taking particular note of its morphology and distribution pattern of the nerve and artery. Forty-nine hands from Korean adult cadavers were used in this study. Parameters with respect to the size, scale, and location of the muscle were measured using a ultrasonography system, digital caliper, and surgical microscope. Histologic sections were obtained to clarify the positional relationship with surrounding tissues. The branches of the ulnar nerve and artery, which supply the muscle, were observed. The minimum and maximum depths in which the palmaris brevis muscle can be existed, including the thickness of the skin (1.9 mm), were 3.7 and 6.9 mm, respectively. It was inserted into the hypothenar fascia and the dermis. The main trunk of the ulnar nerve and artery were positioned under the lateral side of the muscle. The richest arborization of perforating branches of the ulnar nerve and artery was mostly confined to the distal section of the lateral side of the muscle. Our findings provide theoretical evidence of the palmaris brevis muscle for clinical procedures of the hand, such as botulinum toxin injection. Clinicians should pay attention not to injure the ulnar artery under the lateral side of the muscle. Botulinum toxin should be injected at a depth of between approximately 2 and 7 mm, considering the range of depth of the muscle. Clin. Anat. 30:397-403, 2017. © 2017 Wiley Periodicals, Inc.