Objective.This study aims to characterize the motor units (MUs) distribution and recruitment pattern in the spastic and non-spastic bilateral biceps brachii muscles (BBMs) of chronic stroke survivors.Approach.High-density surface electromyography (HD-sEMG) signals were collected from both spastic and non-spastic BBMs of fourteen chronic stroke subjects during isometric elbow flexion at 10%, 30%, 50% and 100% maximal voluntary contractions (MVCs). By combining HD-sEMG decomposition and bioelectrical source imaging, MU innervation zones (MUIZs) of the decomposed MUs were first localized in the 3D space of spastic and non-spastic BBMs. The MU depth defined as the distance between the localized MUIZ and its normal projection on the skin surface was then normalized to the arm radius of each subject and averaged at given contraction level. The averaged MU depth at different contraction levels on a specific arm side (intra-side) and the bilateral depths under a specific contraction level (inter-side) were compared.Main results.The average depth of decomposed MUs increased with the contraction force and significant differences observed between 10% vs 50% (p< 0.0001), 10% vs 100% (p< 0.0001) and 30% vs 100% MVC (p= 0.0017) on the non-spastic side, indicating that larger MUs with higher recruitment threshold locate in deeper muscle regions. In contrast, no force-related difference in MU depth was observed on the spastic side, suggesting a disruption of orderly recruitment of MUs with increase of force level, or the MU denervation and the subsequent collateral reinnervation secondary to upper motor neuron lesions. Inter-side comparison demonstrated significant MU depth difference at 10% (p= 0.0048) and 100% force effort (p= 0.0026).Significance.This study represents the first effort to non-invasively characterize the MU distribution inside spastic and non-spastic bilateral BBM of chronic stroke patients by combining HD-sEMG recording, EMG signal decomposition and bioelectrical source imaging. The findings of this study advances our understanding regarding the neurophysiology of human muscles and the neuromuscular alterations following stroke. It may also offer important MU depth information for botulinum toxin injection in clinical post-stroke spasticity management.