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Background: Climate change resulting in heat stress, is one of the most challenging environmental conditions affecting poultry. Studying thermotolerance is crucial in the prevention of heat stress in chickens because it may lead to the identification of genetic lines that can withstand adverse effects of heat stress. This study aimed at investigating polymorphisms in heat shock protein 70 (HSP70) gene in indigenous chicken ecotypes. We also analyzed the mitochondrial DNA (mtDNA) D-loop of indigenous chickens to assess their origins and genetic diversity. Methods: We collected samples From Turkana basin, Mt. Elgon catchment, Lake Victoria basin and Lamu chicken ecotypes in Kenya. Genomic DNA was extracted from 280 chicken samples. The first 360 bp region of HSP70 and the first 760 bp region of the mtDNA were then amplified via PCR. These were later sequenced using Sanger ABI 3730 method. Results: We reveal through a detailed analysis of the HSP70 gene fragment in 20 indigenous chickens the presence of three HSP70 haplotypes (GC, AC, and AG) and 28 mtDNA haplotypes. Phylogenetic analysis of HSP70 revealed the presence of the ancestral haplotype GC which dominated in Turkana basin ecotype. The 28 mtDNA haplotypes clustered in haplogroups A, B, C, D, E and I. Haplogroup E which has never been reported in commercial chickens dominated in Turkana basin ecotype indicating no admixture with commercial chickens. mtDNA haplogroups were shown to have originated from various parts of South and Southeast Asia. Lack of population structure in indigenous chicken ecotypes could be an indication of genetic admixture. The mtDNA nucleotide and haplotype diversity indices were low for Turkana basin ecotype and high for Lamu ecotype. High HSP70 nucleotide diversity indices were recorded in Turkana basin ecotype, while low values were recorded in Lamu ecotype. Most of the mtDNA genetic variations occurred within individuals for the three hierarchical categories considered while most variations in HSP70 gene occurred within populations. Conclusions: This is the first study to analyze the HSP70 polymorphisms in indigenous chickens in Africa and results obtained should pave the way for further in-depth studies on heat stress.