Pressure pain threshold (PPT) is defined as the minimum force applied which induces pain. This measure has proven to be commonly useful in evaluating tenderness symptom. Our aim was to study the intra-examiner reproducibility of PPT measurement, define cutoffs in normal groups, and compare these results with patients with fibromyalgia (FM). Fifty healthy females, 50 healthy males, and 20 patients with FM participated in the study. PPTs were assessed for 18 specific tender point sites by a dolorimeter. The intra-individual coefficient of variation determined by a test-retest PPT measurement procedure with 3-days interval reached, respectively, 17% and 13% in healthy females and males, versus 24% in patients with FM. PPTs were significantly lower in healthy females than in healthy males (p<0.01). Statistical analysis failed to show any differences between the dominant and nondominant side for both normal groups. PPTs were lower over all examined areas in patients with FM than those obtained in healthy females (p<0.000). Lower cutoff levels were calculated from normal values for all specific tender point sites. On average, 14 tender point sites in patients with FM were under the established lower cutoffs. In conclusion, pressure pain sensitivity was influenced by the anatomical localization of tender point and gender differences. Lowest PPTs were localized in trapezius, occiput, anterior cervical, and second rib. The reduction of total tender point score in patients with FM averaged 60% comparatively with normal values. PPT reproducibility and discrimination between the two groups were optimal for the gluteal and knee sites.