Brown spots on mature leaves from the heading to ripening stages in rice are considered to be lesions induced by heat stress. However, there are few studies of lesions that are induced by heat stress rather than by pathogen infections. To understand the genetic background underlying such lesions, we used the chromosome segment substitution line (CSSL) SL518, derived from a distant cross between rice cultivars Koshihikari (japonica) and Nona Bokra (indica). We observed brown spots on mature leaf blades of the CSSL, although the parents barely showed any spots. Spot formation in SL518 was accelerated by high temperature, suggesting that the candidate gene for spot formation is related to heat stress response. Using progeny derived from a cross between SL518 and Koshihikari, we mapped the causative gene, BROWN-SPOTTED LEAF 1 (BSPL1), on chromosome 5. We speculated that one or more Nona Bokra genes suppress spot formation caused by BSPL1 and identified candidate genomic regions on chromosomes 2 and 9 using a cross between a near-isogenic line for BSPL1 and other CSSLs possessing Nona Bokra segments in the Koshihikari genetic background. In conclusion, our data support the concept that multiple genes are complementarily involved in brown spot formation induced by heat stress and will be useful for cloning of the novel gene(s) related to the spot formation.