Objective: The study aimed to determine the utility of single-fibre electromyography (SFEMG) in the diagnosis of myasthenia gravis (MG) in subjects with a clinical suspicion of the disease., Methods: We performed a prospective, single-blinded study on 100 consecutive patients. SFEMG was not considered a criterion in making the MG diagnosis. For all cases, a different physician than the one performing SFEMG made the diagnosis of MG. All subjects underwent standard SFEMG of a single muscle, the orbicularis oculi., Results: SFEMG was abnormal in 67 of 100 patients. A final diagnosis of definite MG was made in 54 patients (30 men/24 women). SFEMG was positive in 53 of 54 patients diagnosed with MG. The sensitivity of SFEMG in diagnosing MG was 98% (95% CI: 0.94-1.02), while the specificity was 70% (95% CI: 0.54-0.86), with a positive predictive value of 79% (95% CI: 0.74-0.79) and a negative predictive value of 97% (95% CI: 0.94-0.99)., Conclusions: In this cohort of patients, normal SFEMG findings were unlikely to occur in patients with MG., Significance: SFEMG is not a confirmatory test for the diagnosis of MG, but it has a high negative predictive value in identifying patients without MG., (Copyright © 2013 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.)