We have previously shown that neonatal high oxygen (O2) exposure in rats leads to hypertension and vascular dysfunction in adulthood. Pulse-wave velocity (PWV), an indirect measure of vascular biophysical properties (arterial stiffness or distensibility), is a sensitive marker of cardiovascular health. Its measurement in rats is mostly based on invasive hemodynamics measurements, prohibiting longitudinal studies particularly relevant in models of developmental programming of cardiovascular dysfunctions. With this study, we sought (1) to verify the feasibility and validity of measuring of aortic PWV in Sprague-Dawley rats by ultrasound; (2) to use the technique to compare aortic PWV in adult rats exposed to O2 as newborns (80% day 3-10 of life) v. controls; and (3) to develop an algorithm to calculate PWV in a non-invasive manner. We calculated aortic PWV using standard echocardiography and electrocardiogram, and validated the measures with PWV obtained by intraaortic catheters. Aortic full length was measured at sacrifice. PWV was significantly increased in O2 exposed (505 ± 18 cm/s) v. control animals (421 ± 17 cm/s, P < 0.01). With regard to weight, femur length and distance from the manubrium to the anal margin (MA length), the latter showed the best correlation (R = 0.84, P < 0.0001) with full aorta length derived from (L) = 0.339 × (MA length) + 4.281. The current data using echo-Doppler method demonstrated increased aortic stiffness in adult rats exposed to hyperoxia as newborns and suggests that non-invasive longitudinal studies of aortic PWV can be performed using the proposed algorithm for estimation of the full aorta length.