Low temperatures in summer bring about drastic reduction in seed yield of soybean [Glycine max (L.) Merr.]. To identify quantitative trait loci (QTL) associated with chilling tolerance during the reproductive growth in soybean, a recombinant inbred line (RIL) population consisting of 104 F(6)-derived lines was created from a cross between two cultivars, chilling-tolerant Hayahikari and chilling-sensitive Toyomusume. The RIL were genotyped with 181 molecular and phenotypic markers and were scored with regard to chilling tolerance, which was evaluated by comparison of seed-yielding abilities in two artificial climatic environments at chilling and usual temperatures. Three QTL were detected for chilling tolerance in seed-yielding ability. Two of them, qCTTSW 1 and qCTTSW 2, were mapped near QTL for flowering time, and the latter had an epistatic interaction with a marker locus located near another QTL for flowering time, where no significant QTL for chilling tolerance was detected. The analysis of an F(2) population derived from the cross between Hayahikari and an RIL of the Hayahikari genotype at all QTL for flowering time confirmed the effect of the third QTL, qCTTSW 3, on chilling tolerance and suggested that qCTTSW 1 was basically independent of the QTL for flowering time. The findings and QTL found in this study may provide useful information for marker-assisted selection (MAS) and further genetic studies on soybean chilling tolerance.