Objective: The clinical significance of unligated major left internal mammary artery (LIMA)-side branches (SB) remains controversial in patients with previous coronary artery bypass graft (CABG) surgery. The aim of this study was to investigate the clinical significance of unligated major LIMA-SB by using exercise myocardial perfusion imaging (MPI) with single-photon emission tomography. Subjects and Methods: We conducted a retrospective analysis of 2819 consecutive patients who underwent diagnostic angiography. There were 407 CABG patients with LIMA graft. The demographic, laboratory, pre-angiographic stress test and angiographic data of these patients were collected. A subgroup of patients with unligated major LIMA-SB who were referred to angiography with the diagnosis of stable angina pectoris and positive exercise MPI was identified and divided into two groups for comparison: anterior wall vs non-anterior wall ischemia groups. Results: Among 407 patients with LIMA graft, 112 (27.5%) patients were found to have unligated major LIMA-SB. In a subgroup of patients (n=45) with positive exercise MPI and patent LAD-LIMA system with unligated major LIMA-SB, the median values of diameter and length of unligated major LIMA-SB were statistically higher in anterior wall ischemia group (n=24) compared to non-anterior wall ischemia group (1.8mm vs 0.6mm, P<0.001 and 17.0cm vs 8.0cm, P<0.001, respectively). The cut-off values of unligated major LIMA-SB length and diameter were 11cm and 1.3mm respectively. Unligated major LIMA-SB with a length of >= 11.0cm and a diameter of > 1.3cm had 95.8% of sensitivity and 100% of specificity for predicting anterior wall ischemia on exercise MPI. In patients with anterior wall ischemia, summed stress score and summed difference score were improved after percutaneous coil embolization of large unligated major LIMA-SB with 11.0cm length and > 1.3mm diameter. Conclusion: Large unligated major LIMA-SB with 11.0cm length and > 1.3mm diameter seems to be a potential source of ischemia in CABG patients. We suggest that exercise MPI might be a first option noninvasive test in evaluating the clinical significance of unligated major LIMA-SB and the effectiveness of embolization therapy.