Low temperatures after flowering cause seed cracking (SC) in soybean. Previously, we reported that proanthocyanidin accumulation on the dorsal side of the seed coat, controlled by theIlocus, may lead to cracked seeds; and that homozygousIcIcalleles at theIlocus confer SC tolerance in the line Toiku 248. To discover new genes related to SC tolerance, we evaluated the physical and genetic mechanisms of SC tolerance in the cultivar Toyomizuki (genotypeII). Histological and texture analyses of the seed coat revealed that the ability to maintain hardness and flexibility under low temperature, regardless of proanthocyanidin accumulation in the dorsal seed coat, contributes to SC tolerance in Toyomizuki. This indicated that the SC tolerance mechanism differed between Toyomizuki and Toiku 248. A quantitative trait loci (QTL) analysis of recombinant inbred lines revealed a new, stable QTL related to SC tolerance. The relationship between this new QTL, designated asqCS8-2, and SC tolerance was confirmed in residual heterozygous lines. The distance betweenqCS8-2and the previously identified QTLqCS8-1, which is likely theIcallele, was estimated to be 2–3 Mb, so it will be possible to pyramid these regions to develop new cultivars with increased SC tolerance.