PURPOSE: To investigate the three-dimensional (3D), spectral-domain (SD) optical coherence tomography (3D,SD-OCT) imaging of the macular retinal nerve fiber layer (RNFL) in eyes with glaucoma. METHODS: The study included 38 eyes of 38 patients with glaucoma and 38 normal eyes of 38 volunteers. With a 3D raster scan SD-OCT protocol, 512 × 128 axial scans were acquired over a 6-mm(2) area of the macula. Findings on 3D,SD-OCT images were compared with those on color and red-free fundus photographs and time-domain (TD) OCT. RESULTS: Fourteen (30.4%) more RNFL defects were detected on 3D,SD-OCT images than on color fundus photographs. Of these 14, 12 were detected in 10 (90.9%) of 11 eyes with tessellated fundi (P < 0.0001). On 3D,SD-OCT images, complete loss of the RNFL reflectivity was seen in 63.0% of the RNFL defects and thinning of the RNFL in the rest. On TD-OCT cpRNFL analysis, RNFL defects that appeared on 3D,SD-OCT as a complete loss of RNFL reflectivity were detected more often (P = 0.012) than those that appeared as thinning of the RNFL. Inter-rater agreement was better for RNFL defects on 3D,SD-OCT (0.85) than for those on color (0.62-0.64) or red-free (0.68-0.70) fundus photographs. However, 3D,SD-OCT macular RNFL thickness measurements were substantially reproducible but not as reproducible as macular retinal thickness measurements, and neither was as sensitive as TD-OCT cpRNFL thickness measurements for detecting glaucoma. CONCLUSIONS: 3D,SD-OCT imaging of the macular RNFL is an effective means of detecting macular RNFL defects and their severity in eyes with glaucoma.