Here we describe a method for isolate high molecular weight DNA from commercially available frozen bull semen straws. This protocol is based on a salting-out method and uses several commercially available solutions. It consists of several steps: washing of semen, lysis, removal of proteins and precipitation of genomic DNA. This protocol was used to isolate DNA from sixty semen straws, all of which were successfully sequenced using the CLR sequencing mode on the PacBio SequelII platform.