Charcoal rot caused by the fungus, Macrophomina phaseolina, is one of the most important fungal diseases affecting soybean (Glycine max (L.) Merr.) production worldwide, especially under climate change scenarios. The infection events including microsclerotia germination, hyphal penetration, and pathogen colonization into the infected root tissues of two soybean cultivars, Williams (susceptible) and Hadgeston (resistant), with reported differences in the level of resistance to M. phaseolina, were investigated by histological analyses. The soybean plants were infected by immersing the roots in microsclerotia suspension (1 g of microsclerotes in 300 ml of agarose 0.015%). Pathogen penetration took place through the roots epidermal cells 3 days after inoculation. The observations revealed that pathogen's pre-penetration steps, including microsclerotia germination and hyphae development, are not linked to resistance as these events occurred as the same in both cultivars. However, in the post-penetration steps, there was a significant difference between two cultivars in terms of root colonization by M. phaseolina and disintegration of root tissues. The appearance of adventitious roots in the resistant cultivar in response to the pathogen, and infection of secondary roots at the cell differentiation stage as well as the inability of the pathogen to complete its life cycle in the resistant cultivar, are the most significant findings of this study.