PURPOSE: To evaluate intra- and interobserver reproducibility of macular GCC thickness measurement by automated segmentation on the Canon HS-100 SD-OCT (Tokyo, Japan) in normal (N), hypertensive (OHT) and glaucomatous eyes. METHODS: A total of 179 eyes of 93 patients were included: 90 N, 28 OHT and 36 early glaucoma and 25 advanced glaucoma. All patients underwent a complete ophthalmologic exam, central corneal thickness and 24-2 standard automated perimetry (HFA SITA standard). Each of two observers performed three macular acquisitions with the Canon OCT HS-100. Acquisitions were analyzed with the Glaucoma 3D mode, which estimated the macular GCC thickness in global, superior and inferior hemisectors, and in eight separate macular areas. Reproducibility was assessed by intraclass correlation coefficient (ICC), coefficient of variation (CV) and test-retest variability (TRTV) calculated as 1.96 times the standard deviation. RESULTS: Mean GCC thickness was respectively 92.4 µm, 89.0 µm, 80.7 µm and 71.2 µm in N, OHT, early and advanced glaucomatous eyes. In all groups, intra- and interobserver reproducibility ranged respectively for ICC from 89.8 to 99.8% and from 90.2 to 99.4%, for CV from 0.43 to 1.95% and from 0.58 to 2.16% and for TRTV from 0.8 to 3.22 µm and from 1.04 to 3.53 µm. GCC thickness measurements using the new HS-100 SD-OCT were highly reproducible. However, in the advanced glaucoma group, while the reproducibility of GCC thickness measurement is good in the average, superior and inferior hemisectors of the macula, it was slightly less for the paracentral sectors, especially inferior. These sectors correspond generally to the areas most affected by glaucoma. CONCLUSION: The reproducibility of GCC thickness measurements using the new Canon HS-100 SD-OCT is high for normal, OHT, and glaucomatous eyes. It is thus a reliable and reproducible ancillary test available to the clinician for the examination of glaucomatous optic neuropathies.