Three-dimensional (3D) measurement of the spine can provide important information for functional, developmental, diagnostic, and treatment-effect evaluations. However, existing measurement techniques are either 2-dimensional, highly invasive, or involve a high radiation dose, prohibiting their widespread and repeated use in both research and clinical settings. Non-invasive, non-ionizing, 3D measurement of the spine is still beyond the current state-of-the-art. Towards this goal, we developed an intensity-based hierarchical CT-ultrasound registration approach to quantify the 3D positions and orientations of lumbar vertebrae from 3D freehand ultrasound and one-time computed tomography. The method was validated using a human dry bone specimen (T12-L5) and a porcine cadaver (L2-L6) by comparing the registration results with a gold standard fiducial-based registration. Mean (SD) target registration error and percentage of successful registration were 1.2 (0.6) mm and 100% for the human dry bone specimen, and 2.18 (0.82) mm and 92% for the porcine cadaver, indicating that the method is accurate and robust under clinically realistic conditions. Given that the use of ultrasound eliminates ionizing radiation during pose measurements, we believe that the hierarchical CT-ultrasound registration method is an attractive option for quantifying 3D poses of individual vertebra and motion segment, and thus warrants further investigations.