Objective: To explore the myocardial systolic dysfunction in premature ovarian insufficiency (POI) patients with normal left ventricular ejection fraction (LVEF) using echocardiographic layer-specific myocardial strain imaging. Methods: Forty-eight newly diagnosed, untreated POI patients with normal LVEF (POI group) and fifty healthy female volunteers with matching age, height and weight (controls group) were enrolled. Both groups underwent standard transthoracic echocardiography to obtain conventional left ventricular and layer-specific strain parameters, the layer-specific strain parameters including the subendomyocardial global longitudinal strain (GLSendo), the mid-layer myocardial global longitudinal strain (GLSmid), the subepimyocardial global longitudinal strain (GLSepi), the subendomyocardial global circumferential strain (GCSendo), the mid-layer myocardial global circumferential strain (GCSmid), and the subepimyocardial global circumferential strain (GCSepi). Results: The end-diastolic interventricular septal thickness (IVST) in the POI group was significantly thicker than that in the controls group (P=0.008), and The E, E/A, lateral e′ in POI group were significantly lower than those in the controls group (P<0.05). The GLSendo, GLSmid, GLSepi, GCSendo, GCSmid, and GCSepi in the POI group were significant lower than those in the controls group (P＜0.05). Receiver operating characteristic (ROC) curve showed that the area under the curve (AUC) of GLSepi was the largest in diagnosing impaired left ventricular systolic function in POI patients (AUC=0.706, P=0.000). Conclusions: POI patients with normal LVEF may suffer with subclinical left ventricular myocardial systolic dysfunction. The echocardiographic layer-specific myocardial strain could be more sensitive to be used to detect the subclinical impairment of left ventricular systolic function in POI patients.