Myocardial reflectivity, as assessed by calibrated integrated backscatter (cIB) analysis, is a non-invasive surrogate for the amount of left ventricular (LV) fibrosis. The aim of this study was to assess the myocardial reflectivity pattern in patients with heart failure and preserved ejection fraction (HFpEF), and to evaluate its relationship with longitudinal systolic deformation of LV by 2D-speckle tracking echocardiography, and degree of diastolic dysfunction. Transthoracic echocardiography, myocardial Doppler-derived systolic (Sm) and early diastolic velocity (E′), global longitudinal strain (GLS), and tissue characterization by cIB, were obtained in 86 subjects, 46 with HFpEF, and 40 controls. GLS was significantly impaired in HFpEF patients (−15.4 ± 3.5 % vs −21.5 ± 2.9 % in controls; P < 0.0001). Increased myocardial reflectivity, as evidenced by less negative values of cIB, was also found in HFpEF compared to controls (−21.2 ± 4.4 dB vs −25.3 ± 3.9 dB, P < 0.0001). In HFpEF patients, myocardial reflectivity was positively related to GLS (r = 0.68, P < 0.0001), E/E′ ratio (r = 0.38, P = 0.009), and Tau (r = 0.43, P = 0.002), and inversely related to E′ velocity (r = −0.46, P = 0.0012). These associations remained significant after adjustment for age, preload and afterload indices. Patients with HFpEF show changes of LV structure consistent with enhanced fibrosis—as evidenced by increased myocardial reflectivity- which parallel the degree of diastolic dysfunction, and of longitudinal systolic dysfunction.