Although coronary artery disease (CAD) is characterized by epicardial atherosclerosis and microvascular disease, the importance of evaluating microvascular dysfunction has not been sufficiently recognized in clinical practice. We estimated microvascular disease severity by assessing hyperemic microvascular resistance (MVR), as determined by absolute quantification of myocardial blood flow (MBF) with 13N-ammonia positron emission tomography-myocardial perfusion imaging (PET-MPI). We retrospectively collected data for 23 CAD patients who underwent both stress/rest PET-MPI and invasive coronary angiography (CAG) with fractional flow reserve (FFR) measurement. Among 30 vessels for which FFR measurement was performed, 13 had a low FFR (FFR ≤0.75). For each patient, myocardial segments of a standard 17-segment model were assigned to the stenotic myocardial area perfused by the FFR-measured vessel and a reference normal-perfusion area based on PET-MPI and the coronary distribution on CAG. Hyperemic MVR was calculated by using the formula, hyperemic MVR = hyperemic mean blood pressure × FFR/hyperemic MBF of the stenotic vessel. A strong negative correlation was observed between hyperemic MVR and hyperemic MBF in the reference normal-perfusion area (R = -0.758, P<0.001). Microvascular disease severity in chronic CAD can be estimated by hyperemic MBF of the normal-perfusion area with 13N-ammonia PET-MPI.