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Aims Low cardiac iron levels promote heart failure in experimental models. While cardiac iron concentration (CI) is decreased in patients with advanced heart failure with reduced ejection fraction (HFrEF), CI has never been measured in non‐advanced HFrEF. We measured CI in left ventricular (LV) endomyocardial biopsies (EMB) from patients with non‐advanced HFrEF and explored CI association with systemic iron status and disease severity. Methods and results We enrolled 80 consecutive patients with non‐ischaemic HFrEF with New York Heart Association class II or III symptoms and a median (interquartile range) LV ejection fraction of 25 (18–33)%. CI was 304 (262–373) μg/g dry tissue. CI was not related to immunohistological findings or the presence of cardiotropic viral genomes in EMBs and was not related to biomarkers of systemic iron status or anaemia. Patients with CI in the lowest quartile (CIQ1) had lower body mass indices and more often presented with heart failure histories longer than 6 months than patients in the upper three quartiles (CIQ2–4). CIQ1 patients had higher serum N‐terminal pro‐B‐type natriuretic peptide levels than CIQ2–4 patients [3566 (1513–6412) vs. 1542 (526–2811) ng/L; P = 0.005]. CIQ1 patients also had greater LV end‐diastolic (P = 0.001) and end‐systolic diameter indices (P = 0.003) and higher LV end‐diastolic pressures (P = 0.046) than CIQ2–4 patients. Conclusion Low CI is associated with greater disease severity in patients with non‐advanced non‐ischaemic HFrEF. CI is unrelated to systemic iron homeostasis. The prognostic and therapeutic implications of CI measurements in EMBs should be further explored.