Tetrad sterility, in which only clumps of four premature pollen grains are released from anthers, has been observed in some modern potato cultivars. It is a form of cytoplasmic male sterility caused by the cytoplasm derived from the Mexican tetraploid species Solanum stoloniferum Schltdl. & Bouché., an important source of resistance to Potato virus Y in potato breeding. However, since S. stoloniferum is highly polymorphic, the source of tetrad-sterility-causing cytoplasm is unknown among diverse S. stoloniferum accessions. In this study, we directly crossed 24 S. stoloniferum accessions with pollen from 4x S. tuberosum and obtained 39 hybrids from 12 accessions. Nineteen hybrids from six accessions showed tetrad sterility, with either D/γ- or W/γ-type cytoplasm, and were triploid, tetraploid, or hexaploid. The W/γ-type cytoplasm was not necessarily associated with tetrad sterility. Sequence comparisons of 17 mitochondrial genes and their intergenic regions revealed a length polymorphism in the intergenic region between rpl5 and rps10, in which an amplified band of 859 bp was associated with tetrad sterility. This specific cytoplasm causing tetrad sterility is named TSCsto. The 859-bp band will be a useful diagnostic marker for identifying TSCsto in potato breeding.