To facilitate the genetic analysis of quantitative traits and the use of marker-assisted breeding in rice, we developed a novel mapping population consisting of 39 chromosome segment substitution lines (CSSLs). In each line, a different chromosomal segment of the indica cultivar ‘Kasalath’ was substituted in the genetic background of the japonica cultivar ‘Koshihikari’ (Japanese elite cultivar). The substituted chromosome segments in the 39 CSSLs covered most of the genome, except for small regions at the distal end of the short arm of chromosome 8 and at the distal end of the long arm of chromosome 12. To verify the potential advantages of quantitative trait locus (QTL) detection in these CSSLs, we used the CSSLs to locate QTLs for heading date under three different environmental conditions: a natural summer field in Tsukuba, Japan, long-day conditions (14.5-h light), and short-day conditions (10-h light). The results clearly demonstrated that the use of CSSLs enabled to identify a larger number of QTLs than did a BC₁F₃ population derived from the same cross combination. We examined several advantages of the use of CSSLs in terms of genetic analysis, molecular cloning of QTLs, and marker-assisted selection in rice breeding.