Background: This study investigated the value of layer-specific strain analysis by two-dimensional speckle tracking echocardiography (2D-STE) for evaluating left ventricular (LV) systolic function and synchrony in maintenance hemodialysis (MHD) patients. Methods: A total of 34 MHD patients and 35 healthy controls were enrolled in this study. Dynamic images were collected at the LV apical long-axis, the four-and two-chamber, and the LV short-axis views at the basal, middle, and apical segments. The layer-specific speckle tracking (LST) technique was used to analyze the longitudinal strain (LS) and circumferential strain (CS) of LV sub-endocardium, mid-myocardium, sub-epicardium, global longitudinal strain (GLS), global circumferential strain (GCS), the LV 17 segment time to peak LS (TTP), and the peak strain dispersion (PSD). The differences in these parameters were compared between control and MHD groups, and the correlation between PSD and each LS parameter was examined. The receiver operator characteristic (ROC) curve was used to evaluate the efficacy of three myocardial layer LS and CS in the assessment of LV systolic dysfunction in MHD patients. Results: MHD patients had comparable left ventricular ejection fraction (LVEF), but significantly smaller LV GLS, GCS, and three-layer LS and CS compared to the control group. The three myocardial layer LS of the basal segment, middle segment, and apex segment was significantly reduced in the MHD patients compared to the normal subjects, while the three myocardial layer CS of the basal segment, middle segment, and apex segment was significantly reduced in the MHD patients compared to the normal subjects, except for the sub-endocardium of the middle and apex segment. MHD patients had significantly higher TTP of LV 17 segments and PSD compared to controls, and had delayed peak time in most segments. In addition, PSD of MHD patients was positively correlated with sub-endocardial and mid-myocardial LS and GLS, but not with sub-epicardial LS. The area under the curves (AUCs) of sub-endocardial, mid-myocardial, and sub-epicardial LS in MHD patients were 0.894, 0.852, and 0.870, respectively; the AUCs of sub-epicardial, mid-myocardial, and sub-endocardial CS were 0.852, 0.837, and 0.669, respectively. Conclusions: LST may detect early changes of all three-layer LS and CS and PSD in MHD patients, and is therefore a valuable tool to diagnose LV systolic dysfunction in MHD patients.