Semen cryopreservation is an import toll for reproductive management of fish species, which increasingly it is applied in the commercial production of fish. Cryopreservation need of the cryoprotectors agents (CPAs) that may be a complication factor in DNA extraction that can affect the quality of isolated DNA. Genomic DNA constitutes the total genetic information of an organism and it is used for a many finalities, such as to determine gene structure or to detect the presence or absence of genes of interest. However, simple questions to be analyzed become slow to be answered due to troubles in the execution of basic techniques like DNA isolation. This article describes a protocol for the isolation of genomic DNA from fresh and cryopreserved semen.