Abstract: Objective To assess reliability and validity of the three-dimensional (3-D) ultrasound measurement of apical vertebral rotation in patients with adolescent idiopathic scoliosis (AIS). Methods Sixteen female AIS patients were recruited. Ultrasound examinations were performed using a 3-D ultrasound unit with a SonixGPS system, which were compared with magnetic resonance imaging (MRI) examinations conducted on the same day. Two raters performed 3-D ultrasound scanning in supine position on angle measurements for vertebral rotation in the transverse plane three times, respectively. The center of laminae (COL) method was used to measure apical vertebral rotation in the 3-D ultrasound image, compared with the Aaro-Dahlborn results of MRI. Reliability of the 3-D ultrasound measurements was assessed using intra-class correlation coefficients (ICC), mean absolute deviation (MAD), standard deviation (SD) and standard error of measurement (SEM). Validity of the 3-D ultrasound measurements was assessed using paired Student t-tests, Bland-Altman statistics and Pearson correlation coefficients. The level of significance was set as 0.05. Results 3-D ultrasound had high intra- and inter-rater reliabilities (ICC ［2, k］>0.9, P<0.05) for assessing vertebral rotation. There was no significant difference between the Aaro-Dahlborn results in MRI and the COL results in 3-D ultrasound (P>0.05). High consistencies (Bland-Altman) and correlations (Pearson) were demonstrated between the two methods. Conclusion Radiation-free 3-D ultrasound is a reliable and valid method for measuring apical vertebral rotation in the transverse plane of patients with AIS.