This study investigates the effects of radiofrequency ablation (RFA)-created lesions on an explanted human heart in wedge preparation by simultaneous endo and sub-endo optical mapping. The heart in Langendorff perfusion was ablated under 40 W. The ventricle was stained with Vm sensitive dye Di-4-ANBDQPQ and two excitation light bands of different penetration depths were used (red = 660nm, green = 525nm) to perform a conduction velocity (CV) difference analysis for identification of CV alternans. The relative change in fluorescence (ΔF/F) traces were analyzed before and after ablation. Local activation time (LAT) was determined by the 50% approach. Local CV was obtained using the circle method, and RFA created lesions were characterized by examining the CV alternans correlated with transmural heterogeneities. The presence of CV alternans results from reduced excitability in a non-homogeneous lesion consisting of excitable and non-excitable cells. The absence of CV alternans in optical mapping with green light and their presence with deep-red light illustrates incomplete ablation across the ventricular wall or non-homogeneous ablation in the mid-myocardial layer. The presence of an intramural scar impairs the efficacy of the RFA procedure, suggesting a need for alternative ablations strategies.