There is still lack of a simple, accurate, and noninvasive method for rat aortic pulse wave velocity (PWV) measurement, especially the transit distance cannot be accurately measured. Thus, we aimed to derive an equation for aortic transit distance as a function of the nose-to-rump length (L) and to test the hypothesis that aortic PWV measured by new equation combined with Doppler ultrasound (the "equation method") may have stronger correlation with invasive measurements than traditional "body surface method." Two-hundred male Sprague-Dawley (SD) rats (age ranged 5-24 wk) were included in protocol 1, and the aortic transit distances were measured postmortem. In protocol 2, heart-femoral PWV and carotid-femoral PWV were measured by equation method (hfPWVE, cfPWVE) and also by traditional body surface method (hfPWVS, cfPWVS) in another 30 young and 28 old rats. These measurements were then validated against invasively measured hfPWVI and cfPWVI from the same animal. Protocol 1 showed that the heart-femoral transit distance could be calculated by 0.6086 × L - 1.6523, and the carotid-femoral transit distance by 0.4614 × L + 1.8335. In protocol 2, in young rats, the Pearson r between hfPWVE, cfPWVE, hfPWVS, and cfPWVS and their corresponding invasive measurement were 0.8962, 0.8509, 0.8387, and 0.7828, respectively (all P < 0.0001). In the old group, the results were 0.8718, 0.7999, 0.8330, and 0.7112, respectively (all P < 0.0001). The hfPWVE and cfPWVE showed better agreement with hfPWVI and cfPWVI and lower intra- and interobserver variability compared with hfPWVS and cfPWVS in both groups. These findings demonstrate that this novel methodology provides a simple and reliable method for rat noninvasive aortic PWV measurement.NEW & NOTEWORTHY First, when measuring aortic PWV in SD rat models, the heart-femoral transit distance can be estimated by 0.6086 × L - 1.6523, and the carotid-femoral distance transit distance can be estimated by 0.4614 × L + 1.8335, where L (in mm) is nose-to-rump length. Second, this novel methodology for aortic PWV measurement was validated with a closer correlation with the invasive measurements than traditional approach in young and old rats. Third, this study provides a simple and reliable method for rat noninvasive aortic PWV measurement.