Purpose: Macular pigment, composed of lutein, zeaxanthin, and meso-zeaxanthin, is postulated to protect against age-related macular degeneration, likely because of filtering blue light and its antioxidant properties. Macular pigment optical density (MPOD) is reported to be associated with macular function evaluated by visual acuity and multifocal electroretinogram. Given the importance of macular pigment, reliable and accurate measurement methods are important. The main purpose of this study is to determine the reproducibility of MPOD measurement by two-wavelength autofluorescence method using scanning laser ophthalmoscopy. Methods: Sixty-eight eyes of 39 persons were enrolled in the study, including 11 normal eyes, 16 eyes with wet age-related macular degeneration, 16 eyes with dry age-related macular degeneration, 11 eyes with macular edema due to diabetic mellitus, branch retinal vein occlusion or macular telangiectasia, and 14 eyes with tractional maculopathy, including vitreomacular traction, epiretinal membrane, or macular hole. MPOD was measured with a two-wavelength (488 and 514 nm) autofluorescence method with the Spectralis HRA + OCT after pupil dilation. The measurement was repeated for each eye 10 minutes later. The analysis of variance and Bland-Altman plot were used to assess the reproducibility between the two measurements. Results: The mean MPOD at eccentricities of 1 degrees and 2 degrees was 0.36 +/- 0.17 (range: 0.04-0.69) and 0.15 +/- 0.08 (range: -0.03 to 0.35) for the first measurement and 0.35 +/- 0.17 (range: 0.02-0.68) and 0.15 +/- 0.08 (range: -0.01 to 0.33) for the second measurement, respectively. The difference between the 2 measurements was not statistically significant, and the Bland-Altman plot showed 7.4% and 5.9% points outside the 95% limits of agreement, indicating an overall excellent reproducibility. Similarly, there is no significant difference between the first and second measurements of MPOD volume within eccentricities of 1 degrees, 2 degrees, and 6 degrees radius, and the Bland-Altman plot showed 8.8%, 2.9%, and 4.4% points outside the 95% limits of agreement, respectively. The data for the reproducibility did not differ significantly among the various disease and normal eyes. Conclusion: Under routine examination conditions with pupil dilation, MPOD measurement by two-wavelength autofluorescence method showed a high reproducibility.