His bundle is a part of the specialized electrical conduction system that, in the normal or anormal hearts, provides connection between the atrial and ventricular myocardial compartments. The aim of this study was to perform a morphometric analysis of the characteristics of His bundle and its association with predetermined electrophysiological variables in humans, dogs, horses, and pigs. We used five hearts of the species studied. Histological sections of 5 µm thickness were obtained and stained with hematoxylin-eosin and Masson's trichrome. We also used the desmin and PAS method for precise identification of cells. His bundle was longer in horses (2.85 x 0.82 mm) and pigs (1.77 x 0.44 mm) than in dogs (1.53 x 0.26 mm) and humans, which was the shortest (1.06 x 0.23 mm). In His bundle cells, the area and diameters were significantly larger in pigs and horses than in humans (p < 0.001) and dogs (p < 0.001). We have found two patterns of organization of the components of His bundle: Group I, with large cells and a high amount of collagen fibers in ungulates (pigs and horses); group II, with smaller cells and less amount of collagen fibers in humans and dogs. Documenting differences in cell size in His bundle allows us to obtain an additional, alternative identification criterion to commonly used ones such as anatomical location. Morphological characteristics of His bundle and its cells in the different species studied coincide with rapid or slow transmission of the electrical impulse when compared with the predetermined electrophysiological variables.