BACKGROUND: Characterizing mid-myocardial substrate during ventricular tachycardia ablation is challenging. OBJECTIVE: We evaluated how electrode spacing, multipolar (MP) signal annotation, and functional mapping affect the identification of conduction delays over endocardial and mid-myocardial substrates. METHODS: 18 patients with ischemic cardiomyopathy underwent ventricular tachycardia ablation with coregistered magnetic resonance imaging scar models. The substrate was mapped in sinus rhythm (SR) and after a single sensed extrastimulus (SE) from the right ventricle. 12 patients (bipolar [BP] group) were mapped with both short-spline (Oct-S) and long-spline OCTARAY catheters. 6 patients (MP group) were mapped with both the Oct-S and OPTRELL catheters. Points within a 1-cm geodesic distance of magnetic resonance imaging conduction corridors were analyzed for late potentials (LPs), conduction velocity (CV), and anisotropic ratio (AR). RESULTS: Over endocardial substrate, SE mapping increased LP density vs SR for all catheters (long-spline OCTARAY: from 6% to 14%; Oct-S: from 1% to 32%; OPTRELL: from 21% to 45%; P < .001). MP annotation identified more LPs than did BP annotation over endocardial (27% vs 9%; P < .001) and mid-myocardial (41% vs 12%; P < .001) corridors. With BP annotation, median local activation time and CV were similar when stratified by corridor depth (P > .1). With MP annotation, local activation time was delayed by 12 ms per 10% increase in depth in SR and by 7 ms after SE (P < .001), and CV slowed over mid-myocardial corridors (18 cm/s vs 39 cm/s; P < .001). Substrate-adjacent conduction vector heterogeneity was greatest with functional MP mapping (AR, 0.59) and smallest with SR BP mapping (AR, 0.76) (P < .001). CONCLUSION: Functional mapping improved the identification of delayed conduction for all catheters, and MP annotation enhanced the delineation of intramural conduction corridors via the endocardial surface. CLINICAL TRIAL ID: NCT05152784.