The first part of this chapter (Sections 1.1 –1.6.) describes the complete positional cloning process with an emphasis on how multicolor in situ hybridization expedites these studies. Multicolor in situ hybridization is very useful in positional cloning because it maps cloned DNA quickly and is the easiest means to solve positional cloning problems. Applications and insights suggested in this chapter are based on published and ongoing positional cloning projects.