Hypertrophic cardiomyopathy (HCM) patients often develop left--ventricular subendocardial ischaemia, a cause of chest symptoms, despite normal epicardial coronary arteries. The aim of this study was to examine whether ultrasonic tissue characterization or late gadolinium enhancement on cardiac magnetic resonance imaging can detect subendocardial ischaemia in patients with HCM. Subendocardial ischaemia was quantified on exercise Tc-99m tetrofosmin myocardial scintigraphy in 29 non-obstructive HCM patients with asymmetric septal hypertrophy. Ultrasonic tissue characterization using cyclic variation of integrated backscatter (CV-IB) and late gadolinium enhancement on cardiac magnetic resonance imaging were analysed separately in the right halves and the left halves of the ventricular septum in relation to subendocardial ischaemia. Subendocardial ischaemia was identified in 17 (59%) patients. The ratio of CV-IB in the right-to-left halves of the ventricular septum was significantly higher in patients with subendocardial ischaemia (1.19 ± 0.10) than those without (0.84 ± 0.10, P = 0.04). The optimal cutoff for the detection of subendocardial ischaemia was the ratio of CV-IB >1.0, with a sensitivity of 80%, specificity of 71%, and accuracy of 76%. On the other hand, late gadolinium enhancement was not associated with subendocardial ischaemia in our cohort. Ultrasonic tissue characterization using CV-IB separately in the right and left halves of the ventricular septum, but not late gadolinium enhancement on magnetic resonance imaging, provided useful information in detecting subendocardial ischaemia in patients with HCM. Ultrasonic tissue characterization may be useful in selecting patients who will benefit from medications to relieve chest symptoms.