Saprolegnia are parasitic on fish and their eggs and known as the most important pathogens in freshwater fish. Infectious diseases caused by water molds can cause losses of freshwater fish in both nature and commercial fish farms. It is very important to obtain the high quality and effective total DNA to study the mechanism of molecular nosogenesis, identify the species using molecular marker and gene diagnoses.In this study, Saprolegnia parasitica, S. ferax, S. dolica, S. sp(1) and S. sp(2)were used as test materials. The DNA extraction methods of Lysozyme, CTAB, improved CTAB, CH4N2O and SDS were compared. Firstly, the 30 mg (net weight) of freshly subcultured mycelium of five different pathogenetic fish fungi were taken to freeze at - 70 degrees C for 30min in 1.5mL Eppendorf tubes, respectively. Then they were transferred to room temperature and grinded using appropriate abrasive tool. This process was repeated once. Secondly, the trituration of five mycelium were digested by lysozyme digestion, CTAB digestion, improved CTAB digestion, CH4N2O digestion and SDS digestion, respectively, then extracted DNA using relevant processes. The yield and quality of DNA were evaluated with ultraviolet radiation spectrophotometer and PCR of the ITS rDNA. The result of ultraviolet radiation spectrophotometer showed that DNA can be obtained using five different methods and the genomic DNA extracted with improved CTAB method had the best quality and yield, the A(260)/A(280) ratio was between 1.79-1.82 and the concentration of DNA was about 45 mu g/mL. The result of PCR showed that five DNA samples extracted with improved CTAB method can be well amplified and obtained the bright, no tail and trim electrophoresis strip. However, there were unclear or vacant electrophoresis strips in the DNA samples extracted with other methods. Therefore, the DNA extraction method of improved CTAB was the best method to gain the DNA of Saprolegnia for the study of molecular level.