To screen the germplasm with heat tolerance in rice, twenty eight varieties (lines) from 11 countries were selected for evaluation of heat tolerance in this study. The tested rice varieties (lines) at flowering stage were subjected to high temperature treatment in phytotron with temperatures equal to or higher than 35.0 ℃ for 10 hours, and the daily average temperature had been 33.5 ℃ for 7 days. The results indicated that the heat tolerance of the tested varieties (lines) varied considerably. The percentage of spikelet fertility of the tested varieties ranged from 0.6% to 58.7％ and their heat tolerance indexes ranged from 0.01 to 0.85 after high temperature treatment. The indica varieties, “Ganxiangnuo” and “N22” were the most heat-tolerant varieties which had the heat tolerance indexes of 0.84 and 0.85, respectively. Twenty-four SSR and InDel markers that could specifically differentiate indica from japonica were used to analyze the differentiation of the tested materials. The degree of indica-type of the tested materials calculated by the ratio of the number of markers that could generate indica-type band pattern to the total number of markers used was positively correlated with their heat tolerance index (P<0.05), suggesting that indica subspecies was more heat-tolerant than japonica subspecies and there would be more chances to identify heat-tolerant rice germplasm when screening is conducted in indica rice.