Cold tolerance at the seedling stage (CTSS) is an important trait affecting stable rice production in temperate climates and areas of high elevation. In this study, 331 single nucleotide polymorphism (SNP) markers were developed and used along with phenotypic evaluation to identify quantitative trait loci (QTLs) associated with CTSS from a mapping population of 184 F(2) plants derived from a cold tolerant wild rice, W1943 (Oryza rufipogon), and a sensitive indica cultivar, Guang-lu-ai 4 (GLA4). Three QTLs were detected on chromosomes 3, 10 and 11. A major locus, qCtss11 (QTL for cold tolerance at seedling stage), was located on the long arm of chromosome 11 explaining about 40% of the phenotypic variation. Introduction of the W1943 allele of qCtss11 to the GLA4 genetic background increased CTSS. Based on the phenotypic and genotypic assessment of advanced backcross progenies, qCtss11 was dissected as a single Mendelian factor. A high-resolution genetic map was constructed using 23 markers across the qCtss11 locus. As a result, qCtss11 was fine mapped to a 60-kb candidate region defined by marker AK24 and GP0030 on chromosome 11, in which six genes were annotated. Expression and resequence analyses of the six candidates supported the hypothesis that Os11g0615600 and/or Os11g0615900 are causal gene(s) of the CTSS.