ObjectiveThis study aimed to characterize amplitude topographies for masseter motor units (MUs) three-dimensionally, and to assess whether high-density surface electromyography (HDsEMG) is able to detect MU samples that represent the masseter’s entire MU pool. MethodsTen healthy adult volunteers participated in the study, which combined three EMG techniques. A HDsEMG grid covering the entire masseter, and intramuscular fine-wire electrodes were used to obtain two independent MU samples for comparison. The MUs’ amplitude profiles in the dimension of muscle depth were determined using scanning EMG. All data were recorded simultaneously during a low, constant contraction level controlled by 3D force feedback. ResultsThe median medio-lateral diameter of 4.4 mm (range: 1.2–7.9 mm) for MUs detected by HDsEMG did not differ significantly (Mann-Whitney-U test, p = 0.805) from that of 3.9 mm (0.6–8.6 mm) for MUs detected by fine-wire EMG. For individual subjects, the medio-lateral diameters of all HDsEMG-detected MUs spanned 70.5% (19.2–75.1%) of the masseter’s thickness. ConclusionsHDsEMG is able to examine small and large MUs from a great masseter proportion in one single measurement. SignificanceClinical application of HDsEMG might contribute to a better understanding of neuromuscular adaptations in patients with temporomandibular disorders (TMD) and could allow for monitoring treatment effects.