Aim: The purpose of the present study was to determine the characteristics of myocardial damage at different stages of diabetes mellitus (DM) using layer-specific myocardial strain. Material and methods: Thirty six New Zealand white rabbits were randomly divided into either the control group (n = 18) or the DM group (induced with alloxan) (n=18). For the myocardial deformation studies echocardiography and layer-specific strain were performed at baseline and after 3, 6, and 9 months in all of the rabbits. Three-layer longitudinal strain (LS) was calculated in the apical 4-chamber view, and three-layer circumferential strain (CS) in the short-axis view at the level of mitral valve. Layer-specific longitudinal and circumferential strains were assessed from endocardium, mid-myocardium and epicardium. For histomorphological study of the heart structure, the rabbits were sacrificed at 3, 6 and 9 months. Routine hematoxylin and eosin staining was performed. Results: The highest absolute values of left ventricular longitudinal strain (LS) and circumferential strain (CS) were registered in the endocardium and the lowest in the epicardium in both groups. At 3 months, there was no significant difference in three-layer LS and CS (p>0.05), but at 6 months the LS of endocardium (LSendo) and CS of endocardium (CSendo) were lower in the DM group compared with the control group; at 9 months, the rest of the parameters were also decreased (p<0.05). Moreover, in ROC analysis at 6 months LSendo yielded better sensitivity and specificity in the detection of diabetic cardiomyopathy (AUC of LSendo was 0.897 and AUC of CSendo was 0.617). With the progression of untreated diabetes, the histopathological abnormalities intensified gradually beginning at 6 months. Conclusion: The progressive impairments in LV myocardial deformation and structure occurs early in diabetic rabbits, the myocardial damage may be nontransmural, and endocardial function is more susceptible to be affected by DM. Layer-specific myocardial strain echocardiography may identify subtle myocardial dysfunction in the early stages of DM.