To compare choroidal thickness before and after pupil dilation. Macular subfoveal, nasal, temporal and average choroidal thicknesses were measured in one eye of 17 healthy individuals and 40 glaucoma patients using enhanced depth imaging optical coherence tomography before and after pupil dilation. Comparisons were made between pre- and post-dilation measurements, and between normal and glaucomatous eyes. No statistically significant differences were found between pre- and post-dilation choroidal thickness measurements both in normal (p = 0.361 for subfoveal, 0.760 for nasal, 0.941 for temporal, 0.881 for average) and glaucomatous eyes (p = 0.687 for subfoveal, 0.340 for nasal, 0.913 for temporal, and 0.642 for average). After adjusting for age, the comparison between normal and glaucomatous eyes showed no significant differences in measurements both before (p = 0.701-0.907) and after pupil dilation (p = 0.757-0.988). Similar results were obtained for measurements unadjusted for age. Measurements obtained under the two conditions correlated well in normal (r = 0.92-0.97, p < 0.001) and in glaucomatous eyes (r = 0.84-0.98, p < 0.001). Bland-Altman analyses showed good agreements between them in both groups of eyes, with mean difference ranges of 0.43-2.86 and 0.39-3.08 μm between pre- and post-dilation measurements in normal and glaucomatous eyes, respectively. Subfoveal and average choroidal thickness decreased significantly by 2 μm/year. Each millimeter increase in axial length decreased subfoveal choroidal thickness by 16.5 μm and average thickness by 14.1 μm. Macular choroidal thicknesses measured before and after pupil dilation are comparable and may be used interchangeably without significant discrepancies both in normal and glaucomatous eyes.