Multilocus sequence typing (MLST) was first proposed in 1998 as a typing approach that enables the unambiguous characterization of bacterial isolates in a standardized, reproducible, and portable manner using the human pathogen Neisseria meningitidis as the exemplar organism. Since then, the approach has been applied to a large and growing number of organisms by public health laboratories and research institutions. MLST data, shared by investigators over the world via the Internet, have been successfully exploited in applications ranging from molecular epidemiological investigations to population biology and evolutionary analyses. This chapter describes the practical steps in the development and application of an MLST scheme and some of the common tools and techniques used to obtain the maximum benefit from the data. Considerations pertinent to the implementation of high-capacity MLST projects (i.e., those involving thousands of isolates) are discussed.