SummaryScheduled serial endomyocardial biopsies are executed by an internal jugular vein (IJV) or femoral vein (FV) approach to survey acute rejection after heart transplantation (HTx). However, a better approach site is needed. A total of 379 sessions consisting of 329 IJV approaches and 50 FV approaches in 48 HTx recipients executed at 75 ± 127 days (4-1182 days) after HTx between September 2007 and April 2014 at University of Tokyo Hospital were retrospectively analyzed. The IJV approach had shorter operation and radiation exposure times, and a lower dose of radiation exposure and lower usage of contrast agents than the FV approach (all P < 0.001). There were no fatal complications requiring surgical management or resulting in death during all sessions. The IJV approach had less complications than the FV approach (2.7% versus 10.0%, P = 0.011). Among the complications, atrial tachyarrhythmia occurred only with the IJV approach (0.9%), whereas transient ventricular tachyarrhythmia and bundle branch block were more frequently observed in the FV approach (8.0% versus 0.9%, P = 0.042). In conclusion, endomyocardial biopsy from the IJV approach was safer and less invasive than that of the FV approach if we only consider the incidence of atrial tachyarrhythmia. (Int Heart J 2015; 56: 67-72)