Abstract In the present global warming scenario, it is urgent to impart heat tolerance into the popular high yielding rice (Oryza sativa L.) varieties to reduce yield loss to a great extent. Uma (a prevalent red-grained rice variety in Kerala) was crossed with Nagina 22 (N22), the popular donor for heat tolerance. From the seven promising heat-tolerant F3 plants previously identified through marker assisted selection and field screening, F4 lines (58 nos.) were raised and characterised morphologically. Heat tolerance was scored under natural heat stress during hot summer months in field conditions. Among the evaluated F4 progenies, 26 promising plants registered >75% spikelet fertility. Double panicles were observed in some of the tillers of fourteen F4 plants. During the selection with reported polymorphic markers between parents, all the 26 F4 plants produced the corresponding amplicon for RM5749 (linked marker for heat tolerance in the parental cross Uma x N22) and also for RM9, RM201, RM208, RM225, RM242, RM495, RM3586, RM6100, RM6836, and RM26212, corresponding to the heat tolerant parent N22. Genomic analysis revealed 64% recovery of the Uma genome in seven F4 plants, with maximum genomic regions of Uma on the chromosomes 3 and 5. These promising F4 plants could be forwarded to generate stable and high yielding heat tolerant rice varieties. The identified markers could be used for linkage analysis and QTL mapping in future. Keywords: Heat stress; Rice; spikelet fertility; SSR; QTL