Pulsed-field gel electrophoresis (PFGE) can be used to separate the 16 budding yeast chromosomes on the basis of size. Here we describe a detailed, practical protocol that will allow a novice to perform informative PFGE experiments. We first describe the culture of yeast prior to analysis, along with details of embedding cells in agarose before removal of cell walls. We then detail the procedure to remove protein and RNA from chromosomes and how naked chromosomes are loaded into agarose gels before being subjected to electrophoresis. Finally, we describe how the separated chromosomes can be visualized and photographed.