Aims: This study was undertaken to investigate the inter-observer reproducibility of 2D and 3D ultrasound in the measurement of thyroid gland volume. The symmetry of thyroid lobes in healthy subjects was also investigated. Materials and methods: The volume of the left and right lobes of the thyroid gland was measured in 20 healthy subjects (10 men and 10 women) using 2D and 3D ultrasound. On 2D ultrasound, the thyroid lobe volume was calculated by ellipsoid equation (volume = π/6 × craniocaudal × mediolateral × anteroposterior dimensions), whereas 3D ultrasound volumetric measurements were performed with a 3D add-on system. In each subject, the thyroid gland was scanned by two operators to investigate inter-observer variability. Results: There was a moderate agreement between 2D and 3D ultrasound in the measurement of thyroid volume (r = 0.77). 3D ultrasound (90%) had a higher inter-observer reproducibility than 2D ultrasound (85%) in the measurements. About 74% of healthy subjects had the right thyroid lobe larger than the left lobe. Conclusion: 3D ultrasound is useful in the measurement of thyroid volume with a higher reproducibility than 2D ultrasound. Asymmetry of thyroid lobes was noted in healthy subjects. ; School of Optometry