Heat stress adversely affects global bread wheat (Triticum aestivum L.) productivity. This study aimed to uncover the mechanisms underlying heat tolerance in wheat flag leaves and spike tissues. We compared protein abundance in flag leaves and spikes among wheat genotypes with contrasting heat tolerance—two tolerant (RAJ3765 and HD2932 )and two susceptible (HD2329 and HD2733)—under short-term heat stress (32°C) at ear peep. We identified 31 and 60 changes in protein abundance within flag leaves and spike tissues, respectively.