IMPORTANCE Correct attribution of vascular features in optical coherence tomography (OCT) angiography depends on accurate segmentation of retinal layers. OBJECTIVE To evaluate the segmentation of retinal layers among 3 OCT angiography instruments in the central macula, an area where the superficial and deep vascular plexuses terminate. DESIGN, SETTING, AND PARTICIPANTS A retrospective review of a representative OCT angiogram from 1 patient and an evaluation of the vascular pattern in an autopsied eye were conducted at a community retina practice at a university laboratory. A set of 3 × 3-mm scans centered on the fovea using the Cirrus 5000, RTVue XR Avanti, and Triton DRI OCT platforms with default layer segmentations were used to evaluate segmentation accuracy of a normal macula of a white man in his 60s as an emblematic example. A representative histologic section from the central macula of a normal eye was used as an exemplar. MAIN OUTCOMES AND MEASURES Retinal layer segmentation and resultant vascular image compared with vessels as seen in histologic section. RESULTS The segmentation slab designed to isolate the superficial vascular plexus included the deep vascular plexus in the central macula for all 3 instruments. None of the instruments produced segmented regions that followed the relevant anatomic layers correctly. CONCLUSIONS AND RELEVANCE Because of inherent errors in segmentation, studies of the superficial and deep vascular plexuses using manufacturer-recommended default settings are likely to be biased. A proposal for an improved segmentation strategy is presented.