The purpose of this study is to obtain normative values of the masseter muscle of myasthenia gravis (MG) patients and healthy volunteers by single-fiber electromyography (SFEMG). Stimulation of SFEMG in the masseter muscle was studied in 15 healthy volunteers (men 8, women 7; mean age 40.2, range 21-77) and 30 patients affected by MG (men 16, women 14; mean age 42.8, range 12-75). The mean consecutive difference (MCD) of the individual fiber and the mean MCD per study were determined in the normal group. We recommend the upper normal limit for the individual fibers of jitter and the mean MCD per study in the healthy Chinese adults of 33 μs and 22 μs respectively. Furthermore, in the MG group, the percentage of jitter > upper normal limit jitter and the impulse blocking percentage were detected, which were all significantly different compared to the normal control group (P < 0.01). The overall sensitivity was 90%, with the abnormality in 6 of the 9 ocular MG patients and 100% abnormality in the generalized MG patients. The masseter muscle SFEMG has a high degree of sensitivity. The masseter should be considered for SFEMG in the diagnosis of MG, and added routinely to the tested muscles.