Hybrid breakdown (HB) is an important type of post-zygotic reproductive barrier that inhibits hybrid production during the process of cross-breeding. A novel low temperature-dependent HB was identified in a chromosomal segment substitution line (CSSL) library derived from a cross of two rice ( Oryza sativa L. japonica ) cultivars, Yukihikari and Kirara397. A set of weakness symptoms in a target CSSL was observed at 23°C, but was rescued at 27°C and/or 30°C. Genetic analysis of HB using an F 2:3 population of a cross between a target CSSL and Kirara397 found that a recessive temperature sensitive hybrid breakdown1 ( thb1 ) gene from Yukihikari caused HB in the genetic background of Kirara397. Molecular mapping showed that thb1 was located within a 199-kb fragment on chromosome 6. A genetic study of F 2 populations of reciprocal crosses between Yukihikari and Kirara397 confirmed that this HB was induced by the interaction of two recessive genes. These results provide important clues to further dissect the mechanism of generation of a novel temperature sensitive HB in rice intrasubspecific crosses and suggest that these linked markers will useful in rice breeding.