The origin of life on Earth is a profound biological question, with Bacteria and Archaea—the two principal prokaryotic lineages—central to the inquiry. Together, they represent microbial diversity and offer insights into Earth’s earliest biosphere and evolutionary history. Microorganisms are of significant relevance to humanity, not only as disease agents for some infections but also due to their indispensable contributions to ecosystem functioning, primarily because of their involvement in biogeochemical cycling in various habitats. They influence soil fertility, plant growth, and the overall stability of biological communities across different habitats by mediating the turnover of energy and matter through processes such as decomposition, nutrient cycling, and regulating atmospheric gases. The fields of microbial evolution and systematics are mainly concerned with elucidating the origins, diversification, and classification of these two domains of life. These disciplines are fundamental for comprehending the extensive diversity of life on Earth and the evolutionary mechanisms that have shaped it. Notably, horizontal gene transfer, recombination, mutation, and selection are key evolutionary mechanisms driving genetic innovation and ecological differentiation in microbial populations, influencing phylogeny, function, and ecosystem dynamics. Advances in genomics and bioinformatics have transformed microbial systematics by enhancing polyphasic taxonomy through the integration of phenotypic and phylogenetic data, and have also provided valuable tools to gain deep insight into microbial evolution. This chapter examines the evolutionary history of microorganisms in the context of Bacteria and Archaea, the mechanisms underlying their evolution, the modern methodologies employed in microbial systematics, and the broader implications of these studies for science and society.