Cold tolerance at the bud bursting stage is an important target of rice breeding in double-cropping rice region of South China. The achievements have been made in mapping QTL, whereas these QTL have not been effectively used in rice breeding. Identification and pyramiding of QTL are of interest to achieve a breakthrough in rice breeding for cold tolerance at bud bursting stage. In this study, single segment substitution lines (SSSLs) derived from a cross between cold-tolerant japonica variety ‘IR65598-112-2’ and a popular indica variety ‘Huajingxian 74’ were used to detect and pyramid QTL for cold tolerance at the bud bursting stage. Two QTL qCTBB-3 and qCTBB-12 were identified by evaluating the difference of cold tolerance between SSSL and their recurrent parent ‘Huajingxian 74’. The SSSLs carrying qCTBB-3 or qCTBB-12 showed higher seedling survival percentage than that of their recurrent parent ‘Huajingxian 74’ after cold treatment. Through substitution mapping, two linked cold-tolerant QTL (qCTBB-3a and qCTBB-3b) were found in qCTBB-3 region. Furthermore, QTL pyramiding was performed by inter-cross of SSSLs carrying the cold-tolerant QTL (qCTBB-3a/qCTBB-3b, and qCTBB-6 identified in the previous study) and marker-assisted selection (MAS). The lines harboring three QTL showed cumulative effects on cold tolerance. Collectively, by identification of two cold-tolerant QTL and generation of the pyramiding lines with three QTL, this study provided the genes and parental lines in molecular breeding for cold tolerance at the bud bursting stage in rice.