Aims: To investigate whether glutamate-evoked pain in the masseter region differs between three different depths of injection, targeting subcutaneous, muscle, and bone tissues. Methods: A total of 16 healthy volunteers participated and, in a randomized order, received injections of glutamate (1.0 M, 0.2 mL) and isotonic saline (0.9%, 0.2 mL) in the masseter region that targeted subcutaneous, intramuscular, and bone surface tissues. Following injection, pain intensity was measured using electronic visual analog scale (eVAS) and numeric rating scale (NRS) scores of unpleasantness, tiredness, tension, soreness, and stiffness. Pressure pain sensitivity (PPS), pain drawing areas, and McGill Pain Questionnaire (MPQ) scores were also assessed. Repeated-measures analysis of variance, McNemar test, and Tukey post hoc tests were used for statistical analyses. P .05 was considered statistically significant. Results: Overall, subcutaneous injections induced significantly more unpleasantness and pain than intramuscular injections, and PPS scores evoked after glutamate injection at the surface of the bone were significantly higher than after intramuscular glutamate injection. Subcutaneous glutamate injections were more often described as "sharp" and "pinching." Conclusion: The subcutaneous injection was more painful and unpleasant than the intramuscular injection. The glutamate injection at the surface of the bone sensitized the deep pain tissues to pressure stimulation. Clinically, it may be difficult to differentiate between the source or site of pain originating from the masseter region, but the specific quality and word descriptors could assist in differential diagnosis. Keywords: bone surface, glutamate, myofascial pain, subcutaneous, temporomandibular disorders