Since 1998 multilocus sequence typing (MLST) has been widely used for molecular characterization of bacteria and fungi because of its molecular and technical advantages. MLST data are highly variable and informative, universally comparable, easily validated, accessible through the Internet, and transferable across researchers. MLST data are currently used not only in epidemiological investigations at global and local scales but also in studies of pathogen population dynamics, pathogenicity, and molecular evolution. Here, we review several key aspects of MLST molecular design and development in relation to new sequencing technologies; database submission, storing, access, and management; advantages and disadvantages compared to other typing techniques; statistical methods of analysis with special emphasis on coalescent-based approaches; and their application to population dynamics, epidemiological inferences, species diagnosis, and phylogenetics or phylogenomics. The increasing speed, quantity and quality, and reduced cost of gene and genome nucleotide sequencing, together with improved web-based databases, analytical tools, and rising computer power, will lead to a continued expansion in the use of MLST and its integration with cost-effective pathogen genome sequencing. © 2017 Elsevier Inc. All rights reserved.