Technetium-99 m sestamibi (99mTc-MIBI) scintigraphy can identify non-viable left ventricular (LV) myocardium. However, the optimal cut-off value and the details of decreased 99mTc-MIBI uptake of the non-viable LV myocardium in patients with dilated cardiomyopathy (DCM) have not been well established. This study aimed to evaluate the decrease in 99mTc-MIBI uptake in each segment and in the whole LV myocardium, and to determine cut-off values for identifying non-viable LV myocardium in DCM patients. Overall, 53 DCM patients with reduced LV ejection fraction (LVEF ≤ 40%) who underwent 99mTc-MIBI scintigraphy and any optimization of heart failure treatments were evaluated. LV myocardium was classified as viable or non-viable based on the absolute increase in LVEF of ≥ 10% unit leading to an LVEF of > 40% at follow-up, respectively. The decrease in myocardial 99mTc-MIBI uptake in each of the 17 segments was evaluated using three indices determined by different thresholds or standard references: segmental %uptake, rest score, and defect extent. Changes in the whole LV myocardium were evaluated by the minimum %uptake, and the summed rest score (SRS) and extent of LV defect were obtained using summed data of 17 segments. Segmental evaluation indicated a mild decrease in 99mTc-MIBI uptake in 18 patients with viable LV myocardium, whereas focal severe decrease in uptake was observed in patients with non-viable LV myocardium. In the receiver-operating characteristic curve analysis, the cut-off values of minimum %uptake, SRS, and LV defect extent for predicting non-viable LV were 39% (p < 0.01, area under the curve [AUC]: 0.87), 10 (p < 0.01, AUC: 0.91), and 23% (p < 0.01, AUC: 0.92), respectively. In DCM patients, myocardial 99mTc-MIBI %uptake of < 40% indicated non-viable myocardium. The focal and severe decrease in uptake in approximately more than a quarter of the LV myocardium may indicate non-viable LV.