This protocol is used for extraction of high-molecular-weight genomic DNA from yeast cells such as Candida albicans. We have used the protocol successfully for other fungal species such as Emydomyces testavorans that have considerably different incubation conditions and growth kinetics than C. albicans. The resulting DNA is suitable for long-read genome sequencing as well as many other applications. The protocol uses Zymolyase to spheroplast fungal cells, followed by phenol/chloroform extraction and isopropanol precipitation to collect the genomic DNA. The protocol is scaled for cultured yeast cells in a 50-ml volume. Starting with a large number of cells allows DNA yield to be sacrificed at each step in favor of purity and gentle handling of the preparation. The protocol was based on the method from the Laboratory Course Manual for Methods in Yeast Genetics (Cold Spring Harbor Laboratory, 1986; see References). Modification and addition of protocol steps were made over the years, evolving into the protocol presented here which is a standard method in the Hoyer Laboratory.