Purpose: To establish baseline and variability of oxygen tension (PO<sub>2</sub>) measurements in the choroid, retinal arteries, capillaries, and veins of spontaneously breathing anesthetized rats and determine the effect of a moderate surgical procedure on the chorioretinal PO<sub>2</sub>. Methods: Our previously established optical section phosphorescence imaging technique was utilized to measure PO<sub>2</sub> in the chorioretinal vasculatures. Imaging was performed in 29 spontaneously breathing rats under ketamine/xylazine anesthesia. In 7 rats, blood was drawn using a surgically implanted femoral arterial catheter and analyzed to determine the systemic arterial PO<sub>2</sub>. The PO<sub>2</sub> measurements in 22 rats without surgery (group 1) and 7 surgically instrumented rats (group 2) were statistically compared. The intrasubject variability was calculated by the average standard deviation (SD) of repeated measurements. Results: The average systemic arterial PO<sub>2</sub> was 52 ± 7 mm Hg (mean ± SD) in group 2. In group 1, the average PO<sub>2</sub> measurements in the choroid, retinal arteries, capillaries, and veins were 50 ± 11, 40 ± 5, 39 ± 6, and 30 ± 5 mm Hg, respectively. No statistically significant PO<sub>2</sub> differences in any of the chorioretinal vasculatures were found between the two groups (p > 0.4). The intrasubject variability was 3 mm Hg in the choroid, retinal arteries, capillaries, and veins. Conclusions: Chorioretinal PO<sub>2</sub> measurements in spontaneously breathing anesthetized rats have a relatively low variability, indicating that PO<sub>2</sub> changes due to various physiological alterations can be reliably assessed.