Particle motion due to ultrasonic acoustic radiation in a macroscale, multiwavelength acoustic chamber is investigated and compared with available theories. Primary acoustic radiation force theory has been extensively developed to predict single particle motion in a microscale, single-node acoustic chamber/channel. There is a need to investigate the applicability of this theory to macroscale, multiwavelength acoustic channels utilizing the acoustic radiation force for separating polydispersed particles. A particle-tracking velocimetry (PTV) approach for measuring individual particle motion is developed specifically to track particles as they densify at an acoustic pressure node. Particle motion is tracked over the lifetime of their motion to a node. Good agreement between the experimental and theoretical results is observed in the early stages of particle motion, where particles can be considered individually. Only in the densified region of the acoustic pressure node is there some mismatch with theory. The acoustic energy density of the acoustic chamber, a parameter intrinsically associated with the system by the theory, is also determined experimentally for different conditions and shown to be constant for all investigated system settings. The investigation demonstrates the capability of available theory in predicting the motion of polydispersed particles in macroscale, multiwavelength acoustic chambers.