Abstract Introduction Diastolic dysfunction is the earliest cardiac abnormality in β-thalassemia major (β-TM) patients and HFpEF is the predominant cardiac phenotype in symptomatic β-TM patients. Left atrial stiffness index (LASI) has been proved to have prognostic value in HFpEF patients. Purpose In our study we aimed to evaluate LASI in β-TM patients and assess its relation with conventional indices of diastolic function. Methods Sixty β-TM patients and twenty-nine controls were included in this cross-sectional study. Left ventricular ejection fraction (LVEF), early mitral inflow velocity to basal septal early diastolic velocity ratio (E/e'), left atrial volume index (LAVI), tricuspid valve regurgitation velocity (TVR) and left atrial deformation at reservoir phase (LASr) were calculated. T2* was used for myocardial iron load estimation. LASI was calculated as the [E/e’]/[LASr] ratio. Patients with LVEF <50% were excluded. LAVI, E/e’ and TVR were used as criteria of diastolic dysfunction. Results LASI was greater in patients compared to controls (0.30 ± 0.22 vs 0.18 ± 0.06, p = 0.003). Patients with diastolic dysfunction (n = 16) had greater LASI than patients without (n = 20) (0.49 ± 0.35 vs 0.21 ± 0.05, p = 0.001) as well as lower LASr (34.0 ± 15.6% vs 42.1 ± 6.7%, p = 0.043). Patients with indeterminate diastolic status (n = 24) had LASI = 0.26 ± 0.08 and LASr = 45.5 ± 13.6%. No correlation was found between LASI and T2* (p = 0.221). Conclusions β-TM patients with echocardiographic evidence of diastolic dysfunction have increased LASI. Myocardial iron load did not correlate with left atrial stiffness. A significant portion of β-TM patients have indeterminate diastolic function based on conventional criteria and novel indices, such as LASI or LASr, could be useful for reclassification of these particular patients.