The aim of this study is to investigate the effect of Myasthenia Gravis (MG) on the Central Nervous System (CNS) and/or the smooth muscles of the iris through pupillometry. Sixteen recently diagnosed Myasthenic and sixteen non-Myasthenic subjects of matching age and gender underwent a pupillometric study of the effects of single flash stimuli of 24.6 candelas/m2 intensity and 20 msec duration. A significant decrease in Amplitude (p < 0.001), Maximum Constriction Velocity (p < 0.001) and especially Maximum Constriction Acceleration with a perfect discrimination ability (AUC= 1, p < 0.001). was observed in the Myasthenic compared to the non-Myasthenic subjects. In contrast, no significant difference was observed in Baseline Pupil Radius (R1) and 3.5 secs Percentage Recovery-Redilatation (R%) (p = 0.051 and p = 0.517, respectively). Of the parameters that are studied, R1 and R% are governed mainly by the action of the Sympathetic Nervous System (SNS) and the rest by the Parasympathetic Nervous System (ParNS), through Acetylcholine. The analysis of these parameters demonstrates that the SNS remains unaltered while the ParNS may be affected in MG. This post-synaptic cholinergic receptors' deficit may be central, within the CNS, or peripheral, related to the Neuromuscular Junction of the iris' sphincter.