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To determine the prevalence of errors in segmentation of the peripapillary retinal nerve fiber layer (RNFL) and macular ganglion cell complex (GCC) boundary in spectral-domain optical coherence tomography (SDOCT) images, and to identify factors associated with the errors.Peripapillary RNFL circle scans and macular 3-dimensional scans of consecutive cases imaged with SDOCT (RS-3000 Advance; Nidek, Gamagori, Japan) were retrospectively reviewed by a glaucoma specialist. Images with signal strength index (SSI)<6 were excluded. Threshold for segmentation failure was determined as 15 degrees in the RNFL scans and 1/24 of the scanned area in the GCC scans. Relationships between segmentation failure and clinical factors were statistically evaluated with univariable and multivariable analyses.This retrospective cross-sectional study included 207 eyes of 117 subjects (mean age, 58.5±16.5 y). Segmentation failure was found in 20.7% of the peripapillary RNFL scans, 16.6% of the 9 mm GCC scans, and 6.9% of the 6 mm GCC scans in SDOCT images. In multivariable logistic regression analyses, low SSI, large disc area, and disease type significantly correlated with RNFL segmentation failure, whereas SSI was the only baseline factor that was significantly associated with GCC segmentation failure.Although segmentation failure was common in both RNFL and GCC scans, it was less frequently observed in GCC scans. SSI, disc area, and disease type were significantly associated with segmentation failure. Predictive performance of baseline factors for failure was poor, underlining the importance of reviewing raw OCT images before using OCT parameters.