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Heat stress has been one of the major concerns in reducing animal's productivity in tropical, sub-tropical and arid areas. One of the HSPs named Hsp90ab1 protects cells from toxic effects of heat and other stresses. Hsp90ab1 gene has been mapped on BTA 23 and spans nearly 5883 bp consisting of 13 exons and 12 introns. The present investigation was carried out to identify SNPs in the targeted regions of Hsp90ab1 gene in Sahiwal, Karan Fries and Jersey crossbred cows and to analyze the association of genetic variants with respiration rate and rectal temperature. It is observed that cows with CT genotype at 17872061 position in Sahiwal cows was desirable for rectal temperature, whereas, AA genotype at 17872199 position and CC genotype at 17872112 position were desirable for respiration rate in Karan Fries and Jersey crossbred cows respectively.