Aspergillus fumigatus is an opportunistic human pathogenic mould. DNA extraction from this fungus is usually performed by mechanical perturbation of cells as it possesses a rigid and complex cell wall. While this is not problematic for single isolates, it can be time consuming for large numbers of strains by using traditional DNA extraction procedures. Therefore, in this protocol we describe a fast and efficient thermal shock method to release DNA from spores of A. fumigatus and other filamentous fungi without the need of complex extraction methods. This is especially important for high-throughput PCR analyses of mutants such as in 96 or 384 well formats in a very short period of time without any concern of sample cross-contamination. This protocol is currently being used to validate the protein coding gene and non-coding RNA knock-out libraries in A. fumigatus generated in our laboratory and could be used in the future for diagnostics purposes.