Pulsed field gel electrophoresis enables separation of fungal chromosomes up to several megabases and is a worthwhile tool for fungal karyotyping. The germ tube burst method is a technique to separate fungal chromosomes of any size for chromosome number determination as well as in situ hybridization. Here we provide detailed protocols for both complementary methods that have many applications in fungal biology including chromosome size and chromosome number polymorphisms, and in situ localization of genes on chromosomes.