Cold stress is one of the serious abiotic stresses for stable rice production especially in high-latitude temperate region and high-altitude tropical area. Improving cold tolerance at seedling stage led stable seedling growth with yield stability. In this study, QTLs for cold tolerance at seedling stage were identified using the 96 introgression lines (ILs) derived from an inter-specific cross between Hwaseong (Oryza sativa) and Oryza rufipogon. Three QTLs were detected and the O. rufipogon alleles at two QTL (qCTS1 and qCTS12) improved cold tolerance in the Hwaseong genetic background whereas the O. rufipogon allele at qCTS10 on chromosome 10 decreased cold tolerance. Among these three QTLs, a major QTL qCTS12 explained 27.5% of phenotypic variation. Fine-mapping indicated that qCTS12 was different from those QTL reported in previous studies based on the map location suggesting that qCTS12 might be a new allele and is not associated with deleterious genes such fertility reduction. Among the 96 introgression lines, two lines, CR60 and CR61 were selected based on enhanced cold tolerance at seedling stage. qCTS12, therefore, provides a valuable allele for breeding rice with improved cold tolerance.

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