Coccidioides spp. are fungal pathogens present in the arid soil of the American Southwest.This group of pathogenic fungi are responsible for coccidioidomycosis, a disease commonly known as Valley fever.Although Coccidioides infects thousands of people each year, the organism is rather poorly characterized, and little is known about mechanisms of infection.Currently, there is no effective vaccine or treatment against coccidioidomycosis.Understanding the function of an organism’s genes is essential to developing vaccines and treatments because each gene may reveal a vulnerability in the organism which can be targeted with new treatments.Molecular biology is one of the primary tools used to gain an enhanced understanding of how gene function relates to pathogenesis and virulence.Many molecular techniques including DNA sequencing and Southern blotting, require input of large quantities of in-tact genomic DNA.However, Coccidioides spp. cells are notoriously hard to disrupt without vigorous mechanical force which is likely to fragment genomic DNA.This protocol details a phenol chloroform DNA extraction optimized to obtain high yields of unfragmented genomic DNA from Coccidioides spp. using liquid nitrogen grinding to disrupt the cell wall without damaging the DNA inside. Although conventional bead beating and homogenization methods offer a high level of workflow convenience, they are incapable of extracting a large amount of unsheared genomic DNA.The protocol outlined here will provide a detailed workflow for extraction of unfragmented genomic DNA from Coccidioides spp. which is essential for downstream molecular genetics applications such as DNA sequencing and Southern blotting.