Background: Clinical methods examining oxygenation parameters in humans have been used in many different care settings, but concerns have been raised regarding their clinical utility when assessing people with darker skin pigmentation. While saturation values can be crucial in emergency medicine, they are equally valuable in assessing disease mechanisms and monitoring change in disease progression. Retinal pigmentation varies across individuals and hence, can impact on retinal oxygen parameters. The objective of this study was to quantify and eliminate the impact of retinal pigmentation on retinal vessel oxygen saturation parameters measured in the superficial retinal arterioles and venules. Methods: 105 healthy individuals of varying skin colour, iris colour and heritage were included. Following a full eye exam to exclude any ocular abnormality, all participants underwent intraocular pressure, systemic blood pressure measurements and dilated dual wavelength retinal photography. Rotation matrices were employed to minimise the dependency of retinal pigmentation on arterial and venous oxygen saturation measurements determined in a concentric measurement annulus. Results: Retinal oxygen saturation in venules showed a linear correlation with retinal pigmentation (y = 0.34 × x + 38.598), whereas arterial saturation followed a polynomial pattern (y = 0.0089 × x2 + 0.7499 × x + 85.073). Both arterial and venous saturation values were corrected using local fundus pigmentation. Pre-correction retinal arterial and venous oxygen saturation were 89.0% (±13.1) and 43.7% (±11.5), respectively, and post- correction values were 94.8% (±8.7) for arteries and 56.3% (±7.0) veins. Conclusions: When assessing multi-ethnic cohorts, it is important to consider the impact of pigmentation on imaging parameters and to account for it prior to clinical interpretation.