Completion of the fission yeast genome sequence has opened up possibilities for post‐genomic approaches. We have constructed a DNA microarray for genome‐wide gene expression analysis in fission yeast. The microarray contains DNA fragments, PCR‐amplified from a genomic DNA template, that represent > 99% of the 5000 or so annotated fission yeast genes, as well as a number of control sequences. The GenomePRIDE software used attempts to design similarly sized DNA fragments corresponding to gene regions within single exons, near the 3′‐end of genes that lack homology to other fission yeast genes. To validate the design and utility of the array, we studied expression changes after a 2 h temperature shift from 25 °C to 36 °C, conditions widely used when studying temperature‐sensitive mutants. Obligingly, the vast majority of genes do not change more than two‐fold, supporting the widely held view that temperature‐shift experiments specifically reveal phenotypes associated with temperature‐sensitive mutants. However, we did identify a small group of genes that showed a reproducible change in expression. Importantly, most of these corresponded to previously characterized heat‐shock genes, whose expression has been reported to change after more extreme temperature shifts than those used here. We conclude that the DNA microarray represents a useful resource for fission yeast researchers as well as the broader yeast community, since it will facilitate comparison with the distantly related budding yeast, Saccharomyces cerevisiae. To maximize the utility of this resource, the array and its component parts are fully described in On‐line Supplementary Information and are also available commercially. Copyright © 2003 John Wiley & Sons, Ltd.