Alcohol septal ablation (ASA) has been widely used to treat patients with hypertrophic obstructive cardiomyopathy (HOCM). During the routine ASA procedure, it is difficult to detect the septal injury in real‐time. The aim of the present study was to assess myocardial injury during ASA by recording intracoronary electrocardiogram (IC‐ECG). From 2012 to 2015, 31 HOCM patients were treated with ASA, and IC‐ECG was recorded in 21 patients successfully before and after ethanol injection. The elevation of ST‐segment on IC‐ECG after ethanol injection was expressed as its ratio to the level before injection or the absolute increasing value. Blood samples were collected before and after ASA for measuring changes in cardiac biomarkers. The ratio value of ST‐segment elevation was positively correlated with both the amount of ethanol injected (r = 0.645, P = 0.001) and the myocardial injury size (creatine kinase‐MB area under the curve, AUC of CK‐MB) (r = 0.466, P = 0.017). The absolute increment of ST‐segment was also positively associated with both the amount of ethanol (r = 0.665, P = 0.001) and AUC of CK‐MB (0.685, P = 0.001). However, there was no statistical correlation between the reduction of left ventricular outflow tract gradient and ST‐segment elevation. Additionally no severe ASA procedure‐related complications were observed in our patients. In conclusion, myocardial injury induced by ethanol injection can be assessed immediately by ST‐segment elevation on IC‐ECG. Our study is the first to show that IC‐ECG is a useful method for predicting myocardial injury during ASA in real‐time. This article is protected by copyright. All rights reserved.