Abstract Aims Heart failure with preserved ejection fraction (HFpEF), which is caused by wide various conditions, has become a major public health problem. Transthyretin amyloid cardiomyopathy (ATTR‐CM), which is thought to be an underdiagnosed disease, can cause HFpEF. Non‐invasive diagnosis using 99mTechnetium (Tc)‐pyrophosphate (PYP) scintigraphy enables accurate diagnosis of ATTR‐CM. The aim of this study was to clarify the prevalence and characteristics of ATTR‐CM among Japanese patients with HFpEF. Methods and results This study was a multicentre, prospective, observational study conducted in Japan. We enrolled 373 patients with HFpEF [left ventricular (LV) ejection fraction ≥50%] aged ≥65 years who were admitted to the department of cardiology from September 2018 to January 2022. A 99mTc‐PYP scintigraphy scan was performed during admission in all eligible patients. Cardiac 99mTc‐PYP retention was graded according to a previously reported visual scale ranging from 0 to 3 points. The scan was considered positive when it revealed moderate‐to‐severe 99mTc‐PYP uptake (Grade 2–3) in both ventricles. Patients were divided into ATTR‐CM and non‐ATTR‐CM patients according to positive (Grade 2–3) or negative (Grade 0–1) 99mTc‐PYP scintigraphy, respectively. Medical history, blood tests, electrocardiogram, echocardiography, and magnetic resonance imaging in the two groups of patients were compared. Among the 373 patients with HFpEF, 53 patients (14.2%; 95% confidence interval: 10.7–17.7) showed positive uptake on 99mTc‐PYP scintigraphy. An endomyocardial biopsy was performed in 32 patients and confirmed amyloidosis in all cases. There were no significant differences between the two groups in age, severity of heart failure as assessed by the New York Heart Association (NYHA) functional classification, renal function values, left ventricular ejection fraction, and tricuspid regurgitant pressure gradient (ATTR‐CM, n = 53 vs. non‐ATTR‐CM, n = 320). Patients in the ATTR‐CM group had a higher N‐terminal pro‐brain natriuretic peptide level [2314 (1081–3398) vs. 900 (415–1828), P