Air traffic controllers face serious responsibilities and demanding work tasks on duty, which can lead to them experiencing mental fatigue. Due to the adverse effects that fatigue has on air traffic controllers’ performance, it is important to be able to measure their fatigue levels while on duty in order to be able to intervene when the fatigue reaches critical levels. Pupil diameter is one possible mental fatigue measurement, but there are very few studies that have assessed its validity for measuring fatigue in air traffic controllers in the field. This study therefore aimed to assess the criterion-related validity of pupil diameter as a measure of fatigue in air traffic controllers on duty. Reaction time as measured by the psychomotor vigilance test was used as the gold standard fatigue measurement, while controlling for the effect of illumination on pupil diameter. A two-stage hierarchical multiple linear regression analysis was conducted to evaluate the prediction of pupil diameter from reaction time and illuminance. The result of this study indicates that neither RT nor illumination were significant predictors of the pupil diameter of air traffic controllers on duty. Overall, the result of this study indicates that a more complicated analysis with more data points on both illumination and pupil diameter might be necessary to be able to use illumination as a statistically significant predictor of pupil diameter in the field, and that the criterion-related validity of mean pupil diameter as a measurement of mental fatigue in air traffic controllers on duty may be low.