Three-dimensional (3D) soft tissue tracking is of interest for monitoring organ motion during therapy. Our goal is to assess the tracking performance of a curvilinear 3D ultrasound probe in terms of the accuracy and precision of measured displacements. The first aim was to examine the depth dependence of the tracking performance. This is of interest because the spatial resolution varies with distance from the elevational focus and because the curvilinear geometry of the transducer Page 2 of 39 causes the spatial sampling frequency to decrease with depth. Our second aim was to assess tracking performance as a function of the spatial sampling setting (low, medium or high sampling). These settings are incorporated onto 3D ultrasound machines to allow the user to control the trade-off between spatial sampling and temporal resolution. Volume images of a speckle-producing phantom were acquired before and after the probe had been moved by a known displacement (1, 2 or 8 mm).This allowed us to assess the optimum performance of the tracking algorithm, in the absence of motion. 3D speckle tracking was performed using 3D cross-correlation and sub-voxel displacements were estimated. The tracking performance was found to be best for y displacements and poorest for z displacements. In general, the performance decreased with depth, although the nature of the depth dependence was complex.Under certain conditions, the tracking performance was sufficient to be useful for monitoring organ motion. For example, at the highest sampling setting, for a 2 mm displacement, good accuracy and precision (an error and standard deviation of <0.4mm) were observed at all depths and for all directions of displacement. The trade-off between spatial sampling, temporal resolution and size of field of view is discussed.