The polymorphisms of the hsp70 gene have been associated with diverse resistance of heat stress in hens. The aim of the current study was to explore the genetic variation of the hsp70 gene in Layer hens that bred in Iraq. One hundred-fifty Lohmann breed hens aged 12 months were used in this study. Blood samples were collected during the period from 1st September to 31st December 2018 and examined for detection the polymorphism of hsp70 gene. We have detected four main polymorphisms groups in the coding region of hsp70 gene among these layer hens. A significant association between the silent and the missense mutations with the polymorphisms of hsp70 gene in Layer hens was found. There was a high homology of the hsp70 gene sequences that obtained from our local layer hens with the related sequences obtained from different hottest and coldest areas. In conclusion, this study demonstrates that the different mutations (silent and missense) in the coding region of the hsp70 gene of these local Layer hens predict improve birds’ability to the tolerance of stress conditions, and highlights the need of further investigations.