Mutations in the SCN5A gene encoding the cardiac sodium channel protein Nav1.5 are associated with multiple arrhythmia syndromes, including Brugada syndrome, long QT3 syndrome, conduction disease, and sinus node dysfunction. Knowledge of cardiac distribution of Nav1.5 is currently limited but may be crucial for understanding the complex mechanisms involved in sodium channelopathies. Therefore, we investigated Nav1.5 protein expression in various components of the conduction system and transmural layers of the ventricular wall in mouse heart tissue sections. We performed immunohistochemistry and confocal laser scanning microscopy on heart tissue cryosections from adult mice. Nav1.5 showed low to absent expression in the sinoatrial and atrioventricular nodes, whereas the hyperpolarization activated cyclic nucleotide-gated potassium channel 4 (Hcn4) was highly expressed in these areas. In contrast, high expression levels of Nav1.5 were observed in the His bundle and bundle branches (as shown by co-localization with Hcn4). Thus, Hcn4 considered typical for nodal like pacemaker cells and Nav1.5 are not mutually exclusive throughout the entire conduction system. In both ventricles, a transmural gradient was observed, with a low Nav1.5 labeling intensity in the subepicardial layer as compared to midmural; highest Nav1.5 labeling intensity was observed in the subendocardium. The subepicardial layer with low Nav1.5 expression comprised a larger fraction of the total ventricular wall in the right compared to the left ventricle. Overall, the labeling intensity of Nav1.5 within the heart was: His bundle/bundle branches > subendocardium > midmural myocardium >> subepicardium >>> sinoatrial/atrioventricular nodes. The sodium channel protein Nav1.5 shows heterogeneous distribution within the cardiac conduction system, underlying the diverse conduction disorder phenotypes in sodium channelopathies. Decreased Nav1.5 expression in the subepicardium may render the right ventricle in particular more susceptible to sodium channel dysfunction. Thus, transmural heterogeneity in Nav1.5 protein expression may constitute a novel pathophysiological player involved in Brugada syndrome.