Tomographic 3D ultrasound (t3DUS) is a promise imaging technique for quantifying carotid plaque through measuring the degree of stenosis and plaque volume. Carotid plaque volume (CPV) could add benefit in predicting the potential risk of stroke. To assess the reproducibility and accuracy of t3DUS for measuring CPV within the internal carotid artery in patients undergoing carotid endarterectomy. t3DUS was used to obtain CPV in vivo from 25 symptomatic patients prior to surgery. Ex vivo CPV from the carotid endarterectomy specimen was then measured using a validated saline displacement method as a reference standard. CPV for each patient was measured twice using both methods (total n = 50 per technique). Intraclass correlation coefficient (ICC) and Bland-Altman plot were used to establish bias and limit of agreement between CPV measurements. There was an excellent agreement between t3DUS and reference test with respect to measuring CPV with an ICC value of 0.98 (95% confidence interval=0.97-0.99, P < 0.001). Bias in measurements was 0.02 ± 0.11 cm3 (95% limit of agreement=-0.19 to 0.25). Intra-observer agreement of t3DUS CPV measurements was excellent with an ICC value of 0.95 (95% confidence interval=0.92-0.97, P < 0.001). Bias in measurements was 0.004 ± 0.07 cm3 (95% limit of agreement=-0.14 to 0.15). t3DUS is a reproducible imaging method and showed excellent agreement with the reference standard with respect to measuring CPV. These findings suggest that t3DUS has the potential to be a valuable non-invasive tool for assessing carotid plaque burden and predicting the risk of stroke.