Under tropical climate heat stress is a major challenge for livestock production. HSP70.1 is a ubiquitously expressed protein maintaining cellular machinery through proper folding of denatured proteins and prevents cellular apoptosis and protect cell from heat stress. Therefore, present investigation was undertaken to explore genetic variability in HSP70.1 gene in Gangatiri cattle, its comparison with buffalo sequences and differential expression in different season. The allelic variant was identified by sequencing amplified PCR product of HSP70.1 gene by primer walking. Season-wise total RNA samples was prepared for differential expression study. Brilliant SYBR Green QPCR technique was used to study the expression kinetics of this gene. DNA sequencing by primer walking identified four allelic variants in Gangatiri cattle. Sequence alignment study revealed four, six and one substitutions in the 5' untranslated region (5'UTR), coding and 3' untranslated region ((3'UTR) of HSP70.1 gene, respectively. Comparative analysis of HSP70.1 gene revealed that Cattle has shorter 5'UTR and 3' UTR than the buffalo. In Gangatiri cattle, summer season has significantly higher (P ≤ 0.05) expression of HSP70.1 than the spring and winter. The relative expression of HSP70.1 was increased by more than six folds in summer and nearly 1.5 folds higher in winter in comparison to the spring season. Therefore, HSP70.1 may be considered to have a critical role in the development of thermal tolerance in Gangatiri cattle.