Objective This study was to explore a simple and quick polymerase chain reaction (PCR) method for the genotyping of heat shock factor 1(HSF1) knockout mice. Method\ Two pairs of primers were designed to amplify genomic DNA fragment of wild-type HSF1 gene and the same region on HSF1 targeting vector respectively. A gradient PCR strategy was used to test the best annealing temperature. Results\ A 562 bp band was found in wild-type HSF1 mice, a 377 bp band in homozygous mutated HSF1 mice and both bands in heterozygous mice. The genotyping results were completely coincided with those from typical Southern blot. Conclusion \ PCR is a simple, fast and practical method for the genotyping of HSF1 gene knockout mice.