Pulsed field gel electrophoresis (PFGE) uses alternatively oriented pulsed electrical fields to separate large DNA molecules. Here, we describe PFGE protocols and conditions for separating and visualizing chromosomes between 0.5 and 6 Mb (optimal for analyzing the endogenous fission yeast chromosomes of 5.7, 4.6, and 3.5 Mb), and for shorter chromosomal elements of between 50 and 600 kb, such as the 530 kb Ch 16  minichromosome. In addition to determining chromosome size, this technique has a wide range of applications, including determining whether DNA replication or repair is complete, defining the molecular karyotype of cells, analyzing chromosomal rearrangements, assigning genes or constructs to particular chromosomes, and isolating DNA from specific chromosomes.