We aimed to evaluate the diagnostic efficacy of layered velocity vector imaging (VVI)-derived left ventricular (LV) mechanical parameters in the differential diagnosis of primary light-chain cardiac amyloidosis (AL-CA) and hypertrophic cardiomyopathy (HCM). We recruited 35 subjects with histologically-diagnosed AL-CA, 35 subjects with HCM, and 30 age-matched healthy controls. We used conventional echocardiography and electrocardiogram to evaluate general heart function and electrophysiology properties. Furthermore, we applied two-dimensional VVI echocardiography to assess the layered mechanical parameters during systole, including endocardial and epicardial longitudinal strain (ENDO and EPI LSsys), circumferential strain (CSsys), radial strain (RSsys), rotation (ROT) and twist (TWI), in different LV walls and levels. Two groups of patients had similarly elevated LV wall thickness and mild diastolic dysfunction, but normal ejection fraction. ENDO LSsys of three circular LV levels and six LV walls was markedly decreased in AL-CA patients, with the most prominent reduction in the basal level. The reduction of ENDO and EPI LSsys in HCM subjects was less profound, and was restricted to certain LV wall and levels. AL-CA patients had significantly reduced RSsys in the LV basal level compared with control or HCM patients. Two groups of patients exhibited similar reduction in layered regional CSsys, ROT and TWI. ROC analysis revealed that the sensitivity and specificity of basal ENDO LSsys for predicting AL-CA was 86 and 89%. Assessment of layered LSsys of LV walls and levels by VVI appeared to provide a more sensitive and specific diagnostic index for the differential diagnosis of AL-CA from HCM than conventional echocardiography. Future studies are warranted to evaluate its diagnostic efficacy for AL-CA diagnosis in the large population.