High temperature stress is a major obstacle in rice productivity. Considerable progress has been made on studying heat tolerance (HT) at different stages. However， the genetic basis of HT at the booting stage is poorly understood. In this study， we analyzed the morphological features of a heat-sensitive cultivar Sasanishiki under natural high temperature stress at the booting stage. The anthers became smaller and the number， and fertility， of pollen grains were decreased significantly. As a result， there was a dramatic reduction in spikelet fertility. In contrast， the cultivar Habataki showed high HT and normal spikelet fertility under high temperature stress. Additonally， a set of chromosome segment substitution lines， derived from Sasanishiki and Habataki， were evaluated for HT related quantitative trait loci (QTLs) across two environments in the natural field. A total of 12 QTLs associated with HT were detected， of which， 5 were identified in two environments， and 7 in one environment. Furthermore， one of the major-effect QTLs (-) detected on the long arm of chromosome 3， was confirmed using overlapping substituted lines. - was finally mapped between the two markers RM3525 and 3-M95， approximately 2.8 Mb apart. These findings and further gene cloning of - will help us better understand the molecular control of HT in rice， and may contribute to the development of high HT rice varieties.