Aim To analyze the longitudinal peak strain (LPS) of left ventricular subendocardial myocardium, middle myocardium and subepicardial myocardium in patients with diabetes mellitus complicated with microangiopathy by speckle tracking stratified strain technique, so as to evaluate the myocardial function in patients with diabetes mellitus complicated with microangiopathy. Methods 40 patients with type 2 diabetes mellitus (T2DM) were collected and classified according to whether they had microvascular disease:20 cases of T2DM group, 20 cases of T2DM microangiopathy group. 20 cases of healthy volunteers were collected as control group. All subjects underwent conventional echocardiographic examination, and the two-dimensional echocardiographic images of at least three cardiac cycles were collected and stored. The collected two-dimensional dynamic images were imported into the offline analysis software QLab13.0. The global longitudinal strain (GLS) of the left ventricle during systole was obtained by recording the endocardium and epicardium. After that, the left ventricular three-layer myocardium was recorded by stratified strain mapping, and the endocardial longitudinal peak strain (LPSendo), middle myocardial longitudinal peak strain (LPSmid) and epicardial longitudinal peak strain (LPSepi) were obtained. Then the transmural strain pressure difference of left ventricle was obtained from the LPS difference between subendocardial and subepicardial myocardium. Results Compared with control group, GLS in T2DM microangiopathy group significantly decreased by 2.15% (P＜0.05), but there was no significant difference in GLS in T2DM group. Compared with T2DM group, GLS in T2DM microangiopathy group significantly decreased by 1.79% (P＜0.05). Compared with control group, the LPSendo in T2DM group significantly decreased by 3.09% (P＜0.05), but there was no significant difference in the LPSmid and LPSepi in T2DM group (P＞0.05), and LPSendo, LPSmid and LPSepi in T2DM microangiopathy group significantly decreased by 4.20%, 3.38% and 3.16%. Compared with T2DM group, LPSendo, LPSmid and LPSepi in T2DM microangiopathy group respectively decreased by 1.11%, 2.68% and 3.19% (P＜0.05). Compared with control group, ΔLS in T2DM group decreased by 2.59% (P＜0.05), but there was no significant difference in ΔLS in T2DM microangiopathy group, and compared with T2DM microangiopathy group, ΔLS in T2DM group was significantly decreased by 1.89% (P＜0.05). Correlation analysis showed that GLS, LPSendo, LPSmid and LPSepi were negatively correlated with diabetic course (r＝－0.491, －0.546, －0.578 and －0.592, all P＜0.001), there was no linear correlation between ΔLS and diabetic course (r＝0.305, P＞0.05). Conclusion The application of stratified strain technique can sensitively detect the damage of endocardium in diabetic patients and different levels of myocardium in diabetic patients with microangiopathy, which can provide clinical diagnostic basis.