OBJECTIVE : This study describes the use of septal coronary venous mapping to facilitate substrate characterization and ablation of intramural septal ventricular arrhythmia (VA). BACKGROUND : Intramural septal VA represents a challenge for substrate definition and catheter ablation. METHODS : Between 2015 and 2018, 12 patients with structural heart disease, recurrent VA, and suspected intramural septal substrate underwent a septal coronary venous procedure in which mapping was performed by advancement of a wire into the septal perforator branches of the anterior interventricular vein. A total of 5 patients with idiopathic VA were also included as control subjects to compare substrate characteristics. RESULTS : Patients were 63 ± 14 years of age, and 11 (92%) were men. Most patients with structural heart disease had nonischemic cardiomyopathy (83%). Six patients underwent ablation for premature ventricular contractions (PVC) and 6 for ventricular tachycardia. All patients had larger septal unipolar voltage abnormalities than bipolar voltage abnormalities (mean area 35.3 ± 16.8 cm2 vs. 10.7 ± 8.4 cm2, respectively; p = 0.01), Patients with idiopathic VA had normal voltage. Septal coronary venous mapping revealed low-voltage, fractionated, and multicomponent electrograms in sinus rhythm in all patients with substrate compared to that in patients with idiopathic VA (amplitude 0.9 ± 0.9 mV vs. 4.4 ± 3.7 mV, respectively; p = 0.007; and duration 147 ± 48 ms vs. 92 ± 10 ms, respectively; p = 0.03). Ablation targeted early activation, pace map match, and/or good entrainment sites from intraseptal recording. Over a mean follow-up of 339 ± 240 days, the PVC and insertable cardioverter-defibrillator therapies burden were significantly reduced (from a mean of 22 ± 11% to 4 ± 8%; p = 0.005; and a mean 5 ± 2 to 1 ± 1; p = 0.001, respectively). Most patients (80%) with idiopathic VA remained arrhythmia free. CONCLUSIONS : In patients with suspected intramural septal VA, mapping of the septal coronary veins may be helpful to characterize the arrhythmia substrate, identify ablation targets, and guide endocardial ablation.