This study describes the use of septal coronary venous mapping to facilitate substrate characterization and ablation of intramural septal ventricular arrhythmia (VA). Intramural septal VA represents a challenge for substrate definition and catheter ablation. 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. 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. 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. Display omitted