Restriction enzyme analysis of yeast artificial chromosome (YAC) clones in combination with Southern blotting and polymerase chain reaction (PCR) is necessary to establish the integrity of the DNA contained within the YAC clone, as well as obtain information on the integrity of the cloned DNA. Restriction analysis is also a useful tool that allows a direct comparison between endogenous genomic DNA and the DNA fragment present within a YAC clone. This chapter summarizes the techniques of enzyme digestion of YAC DNA and the separation of the DNA fragments by pulse-field gel electrophoresis. The protocols of Southern blotting and PCR used to identify the cloned DNA fragments will also be described.