Low temperature at the booting stage in rice (Oryza sativa L.) can cause male sterility, resulting in yield losses. A set of chromosome segment substitution lines derived from the varieties Sasanishiki (cold-tolerant, ssp. japonica) and Habataki (cold-susceptible, ssp. indica) was used for analysis across two natural, low-temperature environments to study the genetic basis for cold tolerance at the booting stage. Spikelet fertility was used as the evaluation index for cold tolerance identification. Eight quantitative trait loci (QTLs) for cold tolerance were detected, two of which were located on chromosomes 3 (qCTSF3.1 and qCTSF3.2), and the others on chromosomes 4 (qCTSF4), 5 (qCTSF5), 6 (qCTSF6), 7 (qCTSF7), 8 (qCTSF8) and 9 (qCTSF9). The phenotypic variation explained by each QTL ranged from 5.4% to 25.3%. Of the eight QTLs, six (qCTSF3.2, qCTSF5, qCTSF6, qCTSF7, qCTSF8, qCTSF9) were repeatedly detected in two environments. QTLs qCTSF3.1, qCTSF7 and qCTSF9 overlapped with previously reported QTLs. All tolerant alleles for all QTLs were contributed by Sasanishiki.