Low temperature at the booting stage of rice causes male sterility resulting in severe yield loss. Cold tolerance has long been an important objective in rice breeding. We identified a quantitative trait locus (QTL) for cold tolerance on the long arm of chromosome 3 from the cold-tolerant breeding line 'Ukei 840' by using F(2) and BC(1)F(2) populations from crosses between 'Ukei 840' and 'Hitomebore'. The cold tolerance of 'Ukei 840' is derived from the Chinese cultivar 'Lijiangxintuanheigu'. The effect of this QTL on cold tolerance was confirmed by developing 'Hitomebore' chromosome segment substitution lines having 'Lijiangxintuanheigu' alleles on chromosome 3. By producing recombinants in chromosome 3, the QTL region for cold tolerance was delimited to the region of about 1.2-Mb region between RM3719 and RM7000. All lines heterozygous for the QTL showed seed fertilities as low as that of 'Hitomebore', suggesting that the 'Lijiangxintuanheigu' allele for cold tolerance in the QTL region is recessive. Determination of a 1.2-Mb nucleotide sequence of 'Ukei 840' and comparison with the published genomic sequence of 'Nipponbare' showed 254 SNPs, of which 11 were in coding regions of genes, seven in five genes being non-synonymous. SNPs were detected in the 5-kb upstream regions of 89 genes, but no differences of gene expression levels were detected between alleles of these genes. Although further delimitation is required to identify the gene responsible for cold tolerance of 'Lijiangxintuanheigu', SNP markers developed here will be useful for marker-assisted selection in a breeding program using 'Lijiangxintuanheigu' as a donor of cold tolerance.