Aim: To determine if myofascial temporomandibular disorder (TMD) pain patients have elevated interstitial concentrations of glutamate in the masseter muscle. Methods: Thirteen patients (3 men, 10 women) diagnosed with myofascial TMD pain and 10 (2 men, 8 women) age-matched healthy controls participated in a single microdialysis session. Microdialysis was performed in the patients in the most painful point of the masseter muscle, while in the healthy subjects a standardized point in the muscle was chosen. Two microdialysis samples were collected over 40-minute epochs. A blood sample was also taken for analysis of plasma glutamate concentration. Numeric rating scale (NRS) scores of pain intensity and unpleasantness, McGill Pain Questionnaire data, pain drawing areas, pressure pain thresholds, pressure pain tolerances, maximum voluntary bite force, and maximum voluntary mouth opening were collected as secondary measurements. Results: The median concentration of glutamate in the masseter muscle of the myofascial TMD pain patients (7.5 ± 2.6 μM) was significantly higher (P < .023, Mann-Whitney test) than the concentration in healthy controls (0.5 ± 0.4 μM). There were, however, no significant correlations between glutamate concentrations in the masseter muscle and NRS pain scores. Plasma concentrations of glutamate were similar in patients and healthy controls. Conclusions: The present study demonstrates a marked increase in interstitial glutamate concentration in the masseter muscle of myofascial TMD pain patients. These novel findings suggest that peripheral glutamate could be involved in the pathophysiology of myofascial.