Climate change is an imminent threat to livestock production. One adaptation strategy is selection for heat tolerance. While it is established that the ATP1A1 gene and its product play an important role in the response to many stressors, there has been no attempt to characterize the sequence or to perform expression profiling of the gene in production animals. We undertook a field experiment to compare the expression profiles of ATP1A1 in heat-tolerant Vechur and Kasaragod cattle (Bos taurus indicus) with the profile of a heat-susceptible crossbreed (B. t. taurus × B. t. indicus). The cattle were exposed to heat stress while on pasture in the hot summer season. The environmental stress was quantified using the temperature humidity index (THI), while the heat tolerance of each breed was assessed using a heat tolerance coefficient (HTC). The ATP1A1 mRNA of Vechur cattle was amplified from cDNA and sequenced. The HTC varied significantly between the breeds and with time-of-day (p < 0.01). The breed–time-of-day interaction was also significant (p < 0.01). The relative expression of ATP1A1 differed between heat-tolerant and heat-susceptible breeds (p = 0.02). The expression of ATP1A1 at 08:00, 10:00 and 12:00, and the breed–time-of-day interaction, were not significant. The nucleotide sequence of Vechur ATP1A1 showed 99% homology with the B. t. taurus sequence. The protein sequence showed 98% homology with B. t. taurus cattle and with B. grunniens (yak) and 97.7% homology with Ovis aries (sheep). A molecular clock analysis revealed evidence of divergent adaptive evolution of the ATP1A1 gene favoring climate resilience in Vechur cattle. These findings further our knowledge of the relationship between the ATP1A1 gene and heat tolerance in phenotypically incongruent animals. We propose that ATP1A1 could be used in marker assisted selection (MAS) for heat tolerance.