The aim of this study was to determine whether differences exist in oxygen supply to the optic nerve head (ONH) from the retinal and choroidal vascular layers in patients with primary open angle glaucoma (POAG) using multispectral imaging (MSI). This ia an observational, cross-sectional study. Multispectral images were acquired from 38 eyes of 19 patients with POAG, and 42 healthy eyes from 21 matched volunteers with Annidis' RHA multispectral digital ophthalmoscopy. Superficial and deeper oxygen saturation of the optic disc was represented by the mean gray scale values on the retinal and choroidal oxy-deoxy maps, respectively. Statistical analysis was performed to detect differences in ONH oxygen saturation between the 2 groups. Oxygen saturation levels in the eyes of POAG patients with severe glaucoma were compared to those of fellow eyes from the same subjects. Linear correlation analysis was performed to assess the association between ONH oxygen saturation and systemic and ocular parameters. No statistical difference was found in retinal and choroidal oxygen saturation between the POAG and control groups. In the glaucoma patients, retinal oxygen saturation was lower for eyes with worse visual fields than in those with good visual fields (t = 4.009, P = 0.001). In POAG patients, retinal oxygen saturation was dependent on mean defect of visual field and retinal nerve fiber layer thickness (RNFLT) (r = 0.511, 0.504, P = 0.001, 0.001, respectively), whereas the choroid vasculature oxygen saturation was inversely related to RNFLT (r = -0.391, P = 0.015). An age-dependent increase in retinal oxygen saturation was found for both the POAG and control groups ( r= 0.473, 0.410, P = 0.007, 0.003, respectively). MSI revealed a significant correlation between functional and structural impairments in glaucoma and retinal oxygen saturation. MSI could provide objective assessments of perfusion impairments of the glaucomatous ONH. This is a preliminary indication of the effectiveness of MSI for studying POAG.