Background: This study aims to establish a reference for the superior–inferior hemisphere asymmetry in thickness values for all macular layers for the posterior pole algorithm (PPA) available for the Spectralis SD-OCT device. Methods: We examined 300 eyes of 300 healthy Caucasian volunteers aged 18–84 years using the PPA, composed of a grid of 64 (8 × 8) cells, to analyze the thickness asymmetries of the following automatically segmented macular layers: retinal nerve fiber layer (RNFL); ganglion cell layer (GCL); inner plexiform layer (IPL); inner nuclear layer (INL); outer plexiform layer (OPL); outer nuclear layer (ONL); retinal pigment epithelium (RPE); inner retina; outer retina; complete retina. Mean ± standard deviation and the 2.5th and 97.5th percentiles of the thickness asymmetry values were obtained for all the corresponding cells. Results: All the macular layers had significant superior–inferior thickness asymmetries. GCL, IPL, INL, ONL and RPE showed significantly greater thicknesses in the superior than the inferior hemisphere, whereas RNFL and OPL were thicker in the inferior hemisphere. The largest differences between hemispheres were for RNFL and ONL. Conclusions: This is the first normative database of macular thickness asymmetries for the PPA and should be considered to distinguish normal from pathological values when interpreting superior–inferior macular asymmetries.