Aims: To test the hypothesis that there would be no differences in perceived pain intensity and spread and referral of pain evoked by injection of a similar amount of hypertonic saline into 6 different jaw-muscle sites in healthy female subjects. Methods: A total of 15 healthy women participated in 3 experimental sessions separated by 1 week. In a randomized sequence, the deep layers of the masseter, superficial layers of the masseter, anterior temporalis, lateral pterygoid, medial pterygoid, and anterior digastric muscles were injected with 5.8% hypertonic saline (0.2 mL). The subjects rated the perceived intensity of pain on an electronic 0- to 10-cm visual analog scale (VAS). The distribution of pain was drawn by the subjects on anatomical maps of the face, and a Danish version of the McGill Pain Questionnaire (MPQ) was filled out. Results: All injections were associated with moderate to strong pain intensity (mean peak value: 5.6 to 6.4 cm) with no significant differences between muscle sites (analysis of variance [ANOVA]: P = .520). Pain rating indices derived from the MPQ did not suggest significant differences between muscle sites (ANOVA: P = .898). However, the area of perceived pain differed significantly between muscle sites (ANOVA: P = .038) with the greatest area following the injection into the anterior temporalis muscle (Tukey: P .05). On direct inspection, the pain maps appeared quite similar, but a new analysis technique based on a center-of-gravity method revealed significantly different coordinates and length of vectors (ANOVA: P .001) with longer vectors associated with the pain areas in the anterior temporalis muscle compared with the other muscle sites (Tukey: P .05). All muscles were frequently associated with referral of pain to intraoral structures (40% to 87%), but only pain in the anterior digastric muscle was referred to the tip of the tongue (53%). Conclusion: The data suggest no major differences in pain sensitivity between the examined jaw-muscle sites, but pain in the anterior temporalis muscle spreads to a larger area independent of pain intensity. There are subtle but detectable differences in the location and referral of pain patterns between jaw muscles. This will be helpful in the differential diagnosis of myofascial temporomandibular disorder pain. Keywords: jaw-muscle physiology, pain assessment, referred pain, temporomandibular disorder