The fission yeast Schizosaccharomyces octosporus is one of four species in the genus Schizosaccharomyces. Recently released genome sequence data provide useful information for comparative studies. However, Sz. octosporus has not yet been genetically characterized because there have been no heterothallic strains of this species. Here we report the construction of stable heterothallic strains of Sz. octosporus for genetic crosses. First, we continuously observed the mating process of a homothallic strain, yFS286, and determined the mating frequency of Sz. octosporus on various sporulation media. It showed, on average, 30% zygote formation on mating, and a higher frequency of zygotes (43.8 ± 4.7%) on PMG medium. Regardless of sporulation, the number of spores within an ascus was variable. Schizosaccharomyces octosporus forms eight-spored asci, but preferentially produced four-spored asci on MEA or YMoA medium. To obtain heterothallic strains essential for genetic analyses, we isolated spontaneous mutants showing heterothallic-like phenotypes. We also constructed stable heterothallic strains by deleting the silent mat region. As a result, we established the following heterothallic strains, TS162 as h+ and TS150/TS161 as h-, which successfully mated with each other. These genetic tools will be useful for yeast genetics such as molecular cloning, gene complementation tests and tetrad (octad) analysis.