Purpose: We aimed to investigate the association of coronary flow reserve (CFR) with exercise tolerance (ET) in subjects with heart failure with preserved ejection fraction (HFpEF). Materials and Methods: Seventy-seven patients with HFpEF were recruited for the study and compared with 30 healthy matched age and sex controls. All subjects underwent comprehensive echocardiographic evaluation. Exercise tolerance was assessed using 6-minute walking test (6 MWT). CFR was calculated with adenosine stress transthoracic Doppler echocardiography. Results: Out of 77 patients with HFpEF, 51 (66%) had CFR < 2.0. Patients with CFR < 2.0 had lower 6 MWT distance (P < .001), increased E/e' ratio (P < .001), when compared to HFpEF patients with CFR ≥ 2.0 and controls. In subjects with HFpEF, CFR was significantly correlated with 6 MWTD (P < .001) and inversely correlated with E/e" (P < .01). With univariate analysis, the results showed that left ventricular mass index, left atrial volume index, E/e', and CFR were independent predictors for reduced exercise tolerance, whereas at multivariate analysis, reduced CFR was the only independent predictor (P < .001) for both reduced exercise tolerance in patients with HFpEF. Conclusion: The current study demonstrated that reduced CFR was an independent predictor for reduced exercise tolerance in patients with HFpEF. We supposed that microvascular dysfunction has an important role in pathogenesis and clinical course in subjects with HFpEF.