Purpose: The retinal vasculature provides unique in vivo access to the microcirculation and presents the possibility of measuring small artery (retinal) stiffness using pulse wave velocity (PWV). This study investigates whether retinal artery PWV (rPWV) has a blood pressure (BP) dependency. Methods: Fundus videos from eight Sprague-Dawley rats aged 12 weeks were captured (Zeiss fundus microscope with high-speed camera, 125 fps, Optronis, Germany) simultaneously with aortic BP. Retinal artery diameter waveforms at proximal and distal sites were extracted and transit time calculated from the phase delay between frequency components (4–6 Hz, typical heart rate of rats) of the waveforms. rPWV was measured across a physiological range of mean arterial pressure (MAP): baseline (90–110 mmHg); 130 mmHg to baseline following systemic phenylephrine (PE) infusion (30 μg/kg/min); 130 mmHg to baseline during PE infusion with simultaneous inferior vena cava occlusion (VO); 70 mmHg to baseline following systemic sodium nitroprusside infusion; and 70 mmHg to baseline following VO. The correlation between retinal artery rPWV and BP was quantified. Results: There was a significant positive correlation between retinal artery rPWV and MAP as expected (0.19 mm/s/mmHg, R2 = 0.59, p < 0.001). There was a positive correlation between retinal and aortic PWV (R2 = 0.09, p = 0.03). Conclusion: The pressure dependency of the measured rPWV indicates the measure has utility in in vivo quantification of the impact on microvessels of cardiovascular diseases. To elucidate the predictive value of screening rPWV in systemic cardiovascular abnormalities, the relation needs to be investigated in humans.