Objective To study the normal values of the retinal oxygen saturation in Rhesus monkeys and to evaluated repeatability and reproducibility of retinal oxygen saturation measurements. Methods Eighteen adult Rhesus macaque monkeys were included in this experimental study. An Oxymap T1 retinal oximeter (Oxymap, Reykjavik, Iceland) was used to perform oximetry on all subjects. Global arterial (SaO(2)) and venous oxygen saturation (SvO(2)), arteriovenous difference in SO2 were measured. In the first examination, each eye was imaged three times. At the following two examinations, each eye was imaged once. All examinations were finished in one month. P values were calculated to evaluate the difference between the measurements during three visits by performing an ANOVA. Intra-visit and inter-visit intraclass correlation coefficient (ICC) was determined. Results At baseline, the average SaO(2) and SvO(2) were 89.48 +/- 2.64% and 54.85 +/- 2.18%, respectively. The global A-V difference was 34.63 +/- 1.91%. The difference between the three visits was not significant (p>0.05). The highest A-V difference in SO2 and lowest saturations were found in the inferotemporal quadrant. Intra-session and inter-visit repeatability were both high. For all oxygen saturation parameters, the ICC values of the intra-session repeatability ranged between 0.92 and 0.96. As found previously, a relatively high ICC value for intervisit repeatability also was found for all oxygen saturation measurements, ranging between 0.86 and 0.94, with the lowest values in the infero-nasal quadrant. Conclusions Our study is the first to describe retinal SO2 in healthy Rhesus monkeys. In normal monkey eyes, the reproducibility and repeatability of retinal oximetry oxygen saturation measurements were high in the retinal arterioles and venules. Our results support that Oxymap T1 retinal oximetry is a suitable and reliable technique in monkey studies.