The fission yeast Schizosaccharomyces japonicus var. japonicus belong to the genus Schizosaccharomyces, together with Schizosaccharomyces pombe, which has been well studied as a model organism. In contrast, Sz. japonicus is poorly characterized and genetic tools were yet to be developed. We here report the isolation of the heterothallic haploids NIG2017, NIG2025 and NIG2028, which were derivatives of a Sz. japonicus homothallic strain (NIG2008). Based on the genomic sequence of Sz. japonicus, released by the Broad Institute, we found that Sz. japonicus also possesses orthologues of the mating-type genes of Sz. pombe; two mat-M (−) and two mat-P (+) genes. As expected, heterothallic strains were defective in one of the Sz. japonicus mat genes (matsj). We confirmed that NIG2017 and NIG2025 strains only expressed mRNA from the matsj-P genes, while homothallic strains expressed both matsj-M and matsj-P. Although the NIG2028 strain expressed both gene products, matsj-P was found mutated, which may have conferred the heterothallic phenotype of the mutant. Thus, we concluded that these were stable heterothallic strains. We designated NIG2017 and NIG2025 as h+ and NIG 2028 as h−, respectively. We also found additional h− strains (NIG5872 and NIG5873) that arose from the cross between NIG2017 and NIG2028 derivatives. In addition to that, we have constructed a ura4sj-deleted strain and an ade6sj-mutated strain. We used these heterothallic strains and the auxotroph strains to perform spore dissection analysis to determine the genetic distances between several loci, and found that the mating type loci and ade6sj locus were linked to centromeres. Copyright © 2009 John Wiley & Sons, Ltd.