The fission yeast Schizosaccharomyces pombe is a model organism used widely to study various aspects of eukaryotic biology. A collection of heterozygous diploid strains containing individual deletions in nearly all S. pombe genes has been created using a PCR based strategy. However, deletion of some genes has not been possible using this methodology. Here we use an efficient knockout strategy based on plasmids that contain large regions homologous to the target gene to delete an additional 29 genes. The collection of deletion mutants now covers 99% of the fission yeast open reading frames.