Abstract When isolating DNA from fungal cells, the first step in many methods is disruption of cell walls to release the cell contents into the extraction buffer. Often, cell wall disruption is achieved by grinding of the mycelium under liquid nitrogen. There is a large variety in DNA yields and cross contamination can occur due to the cumbersomeness of this method. To shorten and facilitate the DNA isolation procedure, Fusarium cell walls were disrupted by a Polytron homogenizer. The use of the homogenizer for extended periods of time, yielded increasing amounts of DNA but, at the same time, caused increased shearing of DNA. The shearing, however, had no effect on RAPD patterns. In the course of the experiments, it was found that the DNA extraction buffer was capable of releasing substantial quantities of DNA from the Fusarium mycelium (range 0.5–1.6 mg g −1 mycelium), without a separate step for mechanical disruption of cell walls. Both methods described here for isolation of Fusarium DNA, with and without the use of a Polytron homogenizer, substantially reduce the risks of contamination and the risks related to the use of liquid nitrogen. When large quantities of DNA are required, mechanical disruption of the cells using a Polytron homogenizer is suitable. Direct addition of mycelium to the extraction buffer, without a separate step for mechanical cell disruption, is favourable when relatively small amounts of DNA are needed for analysis.