Pulsed-field gel electrophoresis (PFGE) is a general analytical tool to separate large DNA molecules and may therefore be applied to problems from all areas of bacteriology. The genome size of bacteria covers the range of 0.6 to 10 megabase pairs. For genome fingerprinting, the bacterial chromosome is cleaved with a restriction endonuclease that gives a resolvable and informative number of five to one hundred fragments on the PFGE gel. Restriction enzymes are chosen according to GC content, degree of methylation, and codon usage of the respective bacterial genus. Macrorestriction fingerprinting allows the identification of bacterial strains and the distinction between related and unrelated strains. If fragment patterns of several restriction digestions are quantitatively evaluated, strains can be classified according to genetic relatedness at the level of genus, species, and biovar. In particular, members of a clonal lineage can be uncovered. Hence, any problem from applied, environmental, and clinical microbiology may be addressed by PFGE restriction analysis where the spatiotemporal spread of a bacterial clone is of interest. In bacterial genomics, PFGE is employed for the top-down construction of macrorestriction maps of the chromosome which yields data about genome organization, mobile genetic elements, and the arrangement of gene loci and gene families. The genomic diversity of a bacterial species is elucidated by comparative chromosome mapping. Map positions of restriction sites and gene loci of interest serve as landmarks to assess the extent of gross chromosomal modification, namely insertions, deletions and inversions. Intra- and interspecies comparisons of genome organization provide insights into the structure and diversity of bacterial populations and the phylogeny of bacterial taxa.