Purpose: Carotid vessel wall volume (VWV) measurement on three-dimensional ultrasonography (3DUS) outperforms conventional two-dimensional ultrasonography for carotid atherosclerosis evaluation. Although time-saving semi-automated algorithms have been introduced, their clinical availability remains limited due to a lack of validation, particularly an extensive reliability analysis. This study compared inter-observer and intra-observer reliability between manual segmentation and semi-automated segmentation for carotid VWV measurements on 3DUS. Methods: Thirty-one 3DUS volume datasets were prospectively acquired from 20 healthy subjects, aged >18 years, without previous stroke, transient ischemic attack, or cardiovascular disease. Five observers segmented all volume datasets both manually and semi-automatically. The process was repeated five times. Reliability was expressed by the intraclass correlation coefficient, supplemented by the coefficient of variation. Results: Carotid VWV measurements using the common carotid artery (CCA) were more reliable than those using the internal carotid artery (ICA) or external carotid artery (ECA) for both manual and semi -automated segmentation (manual segmentation, CCA: inter-observer, 0.935; intra-observer, 0.934 to 0.966; ICA: inter-observer, 0.784; intra-observer, 0.756 to 0.878; ECA: inter-observer, 0.732; intra-observer, 0.919 to 0.962; semi-automated segmentation, CCA: inter-observer, 0.986; intra-observer, 0.954 to 0.993; ICA: inter-observer, 0.977; intra-observer, 0.958 to 0.978; ECA: inter-observer, 0.966; intra-observer, 0.884 to 0.937). Total carotid VWV measurements by manual (inter-observer, 0.922; intra-observer, 0.927 to 0.961) and semi-automated segmentation (inter-observer, 0.987; intra-observer, 0.968 to 0.989) were highly reliable. Semi-automated segmentation showed higher reliability than manual segmentation for both individual and total carotid VWV measurements. Conclusion: 3DUS carotid VWV measurements of the CCA are more reliable than measurements of the ICA and ECA. Total carotid VWV measurements are highly reliable. Semi-automated segmentation has higher reliability than manual segmentation.