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Aims Coronary microvascular dysfunction (CMD) is related to the pathophysiology, mortality, and morbidity of heart failure with preserved ejection fraction (HFpEF). A novel single-photon emission computed tomography (SPECT) camera with cadmium zinc telluride (CZT) detectors allows for the quantification of absolute myocardial blood flow and myocardial flow reserve (MFR) in patients with coronary artery disease. However, the potential of CZT-SPECT assessing for CMD has never been evaluated in patients with HFpEF. Methods and results The clinical records of 127 consecutive patients who underwent dynamic CZT-SPECT were retrospectively reviewed. Rest and stress scanning were started simultaneously with 3 and 9 MBq/kg of 99mTc-sestamibi administration, respectively. Dynamic CZT-SPECT imaging data were analysed using a net-retention model with commercially available software. Transthoracic echocardiography was performed in all patients. The MFR value was significantly lower in the HFpEF group (mean ± SEM = 2.00 ± 0.097) than that in the non-HFpEF group (mean ± SEM = 2.74 ± 0.14, P = 0.0004). A receiver operating characteristic analysis indicated that if a cut-off value of 2.525 was applied, MFR could efficiently distinguish HFpEF from non-HFpEF. Heart failure with preserved ejection fraction had a consistently low MFR, regardless of the diastolic dysfunction score. Heart failure with preserved ejection fraction patients with MFR values lower than 2.075 had a significantly higher incidence of heart failure exacerbation. Conclusion Myocardial flow reserve assessed by CZT-SPECT was significantly reduced in patients with HFpEF. A lower MFR was associated with a higher hospitalization rate in these patients. Myocardial flow reserve assessed by CZT-SPECT has the potential to predict future adverse events and stratify the severity of disease in patients with HFpEF.